Monoclonal antibodies against influenza virus generated by cyclical administration and uses thereof

ABSTRACT

Provided herein are methods of producing neutralizing monoclonal antibodies, by cyclical immunization, that cross-react with strains of Influenza virus of the same subtype or different subtypes. Also provided herein are compositions comprising such antibodies and methods of using such antibodies to diagnose, prevent or treat Influenza virus disease.

This application claims priority benefit of U.S. provisional applicationNo. 61/181,263, filed May 26, 2009, U.S. provisional application No.61/224,302, filed Jul. 9, 2009, and U.S. provisional application No.61/305,898, filed Feb. 18, 2010, each of which is incorporated herein byreference in its entirety.

This invention was made with United States Government support underaward numbers U01 A170469 and U54 AI057158-06 awarded by the NationalInstitutes of Health (NIH). The United States Government has certainrights in this invention.

1. INTRODUCTION

Provided herein are methods of producing neutralizing monoclonalantibodies, by cyclical administration, that cross-react with strains ofInfluenza virus of the same subtype or different subtypes. Also providedherein are compositions comprising such antibodies and methods of usingsuch antibodies to diagnose, prevent or treat Influenza virus disease.

2. BACKGROUND

Influenza viruses are enveloped RNA viruses that belong to the family ofOrthomyxoviridae (Palese and Shaw (2007) Orthomyxoviridae: The Virusesand Their Replication, 5th ed. Fields' Virology, edited by B. N. Fields,D. M. Knipe and P. M. Howley. Wolters Kluwer Health/Lippincott Williams& Wilkins, Philadelphia, USA, p1647-1689). The natural host of Influenzaviruses are avians, but Influenza viruses (including those of avianorigin) also can infect and cause illness in humans and other animalhosts (canines, pigs, horses, sea mammals, and mustelids). For example,the H5N1 avian Influenza virus circulating in Asia has been found inpigs in China and Indonesia and has also expanded its host range toinclude cats, leopards, and tigers, which generally have not beenconsidered susceptible to Influenza A (CIDRAP—Avian Influenza:Agricultural and Wildlife Considerations). The occurrence of Influenzavirus infections in animals could potentially give rise to humanpandemic Influenza strains.

Influenza A and B viruses are major human pathogens, causing arespiratory disease that ranges in severity from sub-clinical infectionto primary viral pneumonia which can result in death. The clinicaleffects of infection vary with the virulence of the Influenza strain andthe exposure, history, age, and immune status of the host. Thecumulative morbidity and mortality caused by seasonal Influenza issubstantial due to the relatively high rate of infection. In a normalseason, Influenza can cause between 3-5 million cases of severe illnessand is associated with 200,000 to 500,000 deaths worldwide (World HealthOrganization (April, 2009) Influenza (Seasonal) Fact Sheet 211). In theUnited States, Influenza viruses infect an estimated 10-15% of thepopulation (Glezen and Couch R B (1978) Interpandemic Influenza in theHouston area, 1974-76. N Engl J Med 298: 587-592; Fox et al. (1982)Influenza virus infections in Seattle families, 1975-1979. II. Patternof infection in invaded households and relation of age and priorantibody to occurrence of infection and related illness. Am J Epidemiol116: 228-242) and are associated with approximately 30,000 deaths eachyear (Thompson W W et al. (2003) Mortality Associated With Influenza andRespiratory Syncytial Virus in the United States. JAMA 289: 179-186;Belshe (2007) Translational research on vaccines: Influenza as anexample. Clin Pharmacol Ther 82: 745-749).

In addition to annual epidemics, Influenza viruses are the cause ofinfrequent pandemics. For example, Influenza A viruses can causepandemics such as those that occurred in 1918, 1957 and 1968. Due to thelack of pre-formed immunity against the major viral antigen,hemagglutinin (HA), pandemic Influenza viruses can affect greater than50% of the population in a single year and often cause more severedisease than seasonal Influenza viruses. A stark example is the pandemicof 1918, in which an estimated 50-100 million people were killed(Johnson and Mueller (2002) Updating the Accounts: Global Mortality ofthe 1918-1920 “Spanish” Influenza Pandemic Bulletin of the History ofMedicine 76: 105-115). Since the emergence of the highly pathogenicavian H5N1 Influenza virus in the late 1990s (Claas et al. (1998) HumanInfluenza A H5N1 virus related to a highly pathogenic avian Influenzavirus. Lancet 351: 472-7), there have been concerns that the virus maybecome transmissible between humans and cause a major pandemic.

An effective way to protect against Influenza virus infection is throughvaccination; however, current vaccination approaches rely on achieving agood match between circulating strains and the isolates included in thevaccine formulation. Such a match is often difficult to attain due to acombination of factors. First, Influenza viruses are constantlyundergoing change: every 3-5 years the predominant strain of Influenza Avirus is replaced by a variant that has undergone sufficient antigenicdrift to evade existing antibody responses. Isolates to be included invaccine preparations must therefore be selected each year based on theintensive surveillance efforts of the World Health Organization (WHO)collaborating centers. Second, to allow sufficient time for vaccinemanufacture and distribution, strains must be selected approximately sixmonths prior to the initiation of the Influenza season. Occasionally,the predictions of the vaccine strain selection committee areinaccurate, resulting in a substantial drop in the efficacy ofvaccination.

The possibility of a novel subtype of Influenza A virus entering thehuman population also presents a significant challenge to currentvaccination strategies. Since it is impossible to predict what subtypeand strain of Influenza virus will cause the next pandemic, current,strain-specific approaches cannot be used to prepare a pandemicInfluenza vaccine.

3. SUMMARY

Provided herein are methods for generating monoclonal antibodies thatbind to an Influenza virus antigen. Such monoclonal antibodies may bindto an Influenza virus antigen on an Influenza virus particle, e.g.,hemagglutinin (HA). In another specific embodiment, the monoclonalantibodies bind to an Influenza virus particle. In another specificembodiment, the monoclonal antibodies selectively bind to hemagglutininexpressed by one, two, three or more strains of Influenza virus relativeto a non-Influenza virus hemagglutinin antigen as assessed by techniquesknown in the art, e.g., ELISA, Western blot, FACs or BIACore. In otherwords, the monoclonal antibodies bind to hemagglutinin expressed by one,two, three or more strains of Influenza virus with a higher affinitythan a non-Influenza virus hemagglutinin antigen as assessed bytechniques known in the art, e.g., ELISA, Western blot, FACs or BIACore.In a specific embodiment, the monoclonal antibody binds to andneutralizes two or more strains of Influenza viruses that expressantigenically distinct HA. In another embodiment, the monoclonalantibody binds to and neutralizes two or more strains of an Influenza Avirus hemagglutinin (HA) subtype. In another embodiment, the monoclonalantibody binds to and neutralizes strains of Influenza A virus of two ormore HA subtypes. In another specific embodiment, the monoclonalantibody binds to the long alpha-helix of the HA2 region of an Influenzavirus.

In one aspect, a method for generating a monoclonal antibody that bindsto and neutralizes two or more strains of Influenza viruses that expressantigenically distinct HA is provided. In a specific embodiment, amethod for generating a monoclonal antibody that binds to andneutralizes two or more strains of Influenza viruses that expressantigenically distinct HA comprises administering two, three, four ormore immunogenic compositions to a non-human subject with theadministration of each immunogenic composition separated by a certainamount of time, and generating B-cell hybridomas from the subject thatexpress a monoclonal antibody that binds to and neutralizes two or morestrains of an Influenza virus that express antigenically distinct HA(e.g., two or more strains of an Influenza A virus subtype or two ormore strains from distinct Influenza A subtypes), wherein eachimmunogenic composition comprises an inactivated Influenza virus, anattenuated Influenza virus, a live Influenza virus other than anattenuated Influenza virus (e.g., naturally occurring Influenza virus),an antigen derived or obtained from an Influenza virus (e.g., HA), or anucleic acid encoding an antigen derived or obtained from an Influenzavirus, and wherein one immunogenic composition differs from anotherimmunogenic composition in that the Influenza virus, or the Influenzavirus from which the antigen or fragment thereof or the nucleic acidencoding the antigen or fragment thereof is derived or obtained areantigenically distinct. In specific embodiments, the method comprisesselecting for B-cell hybridoma clones that express a monoclonal antibodythat binds to and neutralizes two or more strains of an Influenza viruswhich express antigenically distinct HA. In particular embodiments, thetwo or more strains of Influenza virus which express antigenicallydistinct HA are two or more strains of an Influenza A virus subtype. Incertain aspects, the methods for producing a monoclonal antibody thatbinds to two or more strains of Influenza virus which expressantigenically distinct HA can be used to produce hybridomas that expresssuch an antibody.

In one embodiment, a method for generating a monoclonal antibody thatbinds to and neutralizes two or more strains of an Influenza A virus HAsubtype comprises: (i) administering to a non-human subject a firstimmunogenic composition comprising an inactivated first Influenza virus,an attenuated first Influenza virus, a live first Influenza virus otherthan an attenuated Influenza virus, an HA derived or obtained from afirst Influenza virus or fragment thereof, or a nucleic acid encoding anHA derived or obtained from a first Influenza virus or fragment thereof;(ii) after a first period of time, administering to the subject a secondimmunogenic composition comprising an inactivated second Influenzavirus, an attenuated second Influenza virus, a live second Influenzavirus other than an attenuated Influenza virus, an HA derived orobtained from a second Influenza virus or fragment thereof, or a nucleicacid encoding an HA derived or obtained from a second Influenza virus orfragment thereof, wherein the second Influenza virus is antigenicallydistinct from the first Influenza virus; and (iii) after a second periodof time, generating B-cell hybridomas from the subject that express amonoclonal antibody that binds to and neutralizes two or more strains ofan Influenza A virus HA subtype. In certain embodiments, the methodcomprises selecting for clones that express a monoclonal antibody thatbinds to and neutralizes two or more strains of an Influenza A virussubtype. In certain embodiments, the method further comprises isolatingthe monoclonal antibody. In some embodiments, the method furthercomprises screening for the cross-reactive neutralizing monoclonalantibody. In specific embodiments, the Influenza A virus subtype is H3.In specific embodiments, the Influenza A virus is a Group 2 Influenzavirus (e.g., an Influenza virus that is an H4, H14, H3, H15, H7, or H10subtype). In specific embodiments, the Influenza A virus is a Group 1Influenza virus (e.g., an Influenza virus that is an H1 subtype such asA/South Carolina/1918 (H1), A/USSR/92/77 (H1), A/California/04/09 (H1),or A/Brisbane/59/07-like (H1).

In another embodiment, a method for generating a monoclonal antibodythat binds to and neutralizes two or more strains of an Influenza Avirus HA subtype comprises: (i) administering to a non-human subject afirst immunogenic composition comprising an inactivated first Influenzavirus, an attenuated first Influenza virus, a live first Influenza virusother than an attenuated Influenza virus, an HA derived or obtained froma first Influenza virus or fragment thereof, or a nucleic acid encodingan HA derived or obtained from a first Influenza virus or fragmentthereof; (ii) after a first period of time, administering to the subjecta second immunogenic composition comprising an inactivated secondInfluenza virus, an attenuated second Influenza virus, a live secondInfluenza virus other than an attenuated Influenza virus, an HA derivedor obtained from a second Influenza virus or fragment thereof, or anucleic acid encoding an HA derived or obtained from a second Influenzavirus or fragment thereof, wherein the second Influenza virus isantigenically distinct from the first Influenza virus; (iii) after asecond period of time, administering to the subject a third immunogeniccomposition comprising an inactivated third Influenza virus, anattenuated third Influenza virus, a live third Influenza virus otherthan an attenuated Influenza virus, an HA derived or obtained from athird Influenza virus or fragment thereof, or a nucleic acid encoding anHA derived or obtained from a third Influenza virus or fragment thereof,wherein the third Influenza virus is antigenically distinct from thefirst and the second Influenza viruses; and (iv) after a third period oftime, generating B-cell hybridomas from the subject and furtherselecting for hybridoma clones that express a monoclonal antibody thatbinds to and neutralizes two or more strains of an Influenza A virus HAsubtype. In certain embodiments, the method comprises selecting forhybridoma clones that express a monoclonal antibody that binds to andneutralizes two or more strains of an Influenza A virus subtype. In someembodiments, the method further comprises isolating the monoclonalantibody. In other embodiments, the method further comprises screeningfor the cross-reactive neutralizing monoclonal antibody. In specificembodiments, the Influenza A virus subtype is H3. In specificembodiments, the Influenza A virus is characterized as a Group 2Influenza virus (e.g., an Influenza virus that is an H4, H14, H3, H15,H17, or H10 subtype). In specific embodiments, the Influenza A virus isa Group 1 Influenza virus (e.g., an Influenza virus that is an H1subtype such as A/South Carolina/1918 (H1), A/USSR/92/77 (H1),A/California/04/09 (H1), or A/Brisbane/59/07-like (H1).

In another embodiment, a method for generating a monoclonal antibodythat binds to and neutralizes two or more strains of an Influenza Avirus HA subtype comprises: (i) administering to a non-human subject afirst immunogenic composition comprising an inactivated first Influenzavirus, an attenuated first Influenza virus, a live first Influenza virusother than an attenuated Influenza virus, an HA derived or obtained froma first Influenza virus or fragment thereof, or a nucleic acid encodingan HA derived or obtained from a first Influenza virus or fragmentthereof; (ii) after a first period of time, administering to the subjecta second immunogenic composition comprising an inactivated secondInfluenza virus, an attenuated second Influenza virus, a live secondInfluenza virus other than an attenuated Influenza virus, an HA derivedor obtained from a second Influenza virus or fragment thereof, or anucleic acid encoding an HA derived or obtained from a second Influenzavirus or fragment thereof, wherein the second Influenza virus isantigenically distinct from the first Influenza virus; (iii) after asecond period of time, administering to the subject a third immunogeniccomposition comprising an inactivated third Influenza virus, anattenuated third Influenza virus, a live third Influenza virus otherthan an attenuated Influenza virus, an HA derived or obtained from athird Influenza virus or fragment thereof, or a nucleic acid encoding anHA derived or obtained from a third Influenza virus or fragment thereof,wherein the third Influenza virus is antigenically distinct from thefirst and the second Influenza viruses; (iv) after a third period oftime, administering to the subject a fourth immunogenic compositioncomprising an inactivated fourth Influenza virus, an attenuated fourthInfluenza virus, a live fourth Influenza virus other than an attenuatedInfluenza virus, an HA derived or obtained from a fourth Influenza virusor fragment thereof, or a nucleic acid encoding an HA derived orobtained from a fourth Influenza virus or fragment thereof, wherein thefourth Influenza virus is antigenically distinct from the first, secondand third Influenza viruses; and (v) after a fourth period of time,generating B-cell hybridomas from the subject that express a monoclonalantibody that binds to and neutralizes two or more strains of anInfluenza A virus HA subtype. In certain embodiments, the method furthercomprises isolating the monoclonal antibody. In certain embodiments, themethod comprises selecting hybridoma clones that express a monoclonalantibody that binds to and neutralizes two or more strains of anInfluenza A virus subtype. In some embodiments, the method furthercomprises screening for the cross-reactive neutralizing monoclonalantibody. In specific embodiments, the Influenza A virus ischaracterized as a Group 2 Influenza virus (e.g., an Influenza virusthat is an H4, H14, H3, H15, H17, or H10 subtype). In specificembodiments, the Influenza A virus subtype is H3. In specificembodiments, the first Influenza virus is A/Hong Kong/1/1968, the secondInfluenza virus is A/Alabama/1/1981, the third Influenza virus isA/Beijing/47/1992, and the fourth Influenza virus is A/Wyoming/3/2003.In specific embodiments, the Influenza A virus is a Group 1 Influenzavirus (e.g., an Influenza virus that is an H1 (H1a/H1b) or H9 subtype).In specific embodiments, the Influenza A virus subtype is H1. Inspecific embodiments, the first Influenza virus is A/South Carolina/1918(H1), the second Influenza virus is A/USSR/92/77 (H1), the thirdInfluenza virus is A/California/04/09 (H1), and the fourth Influenzavirus is A/Brisbane/59/07-like (H1).

In another aspect, a method for generating a monoclonal antibody thatbinds to and neutralizes strains of two or more Influenza A virus HAsubtypes is provided. In a specific embodiment, a method for generatinga monoclonal antibody that binds to and neutralizes strains of two ormore Influenza A virus HA subtypes comprises administering two, three,four or more immunogenic compositions to a non-human subject with theadministration of each immunogenic composition separated by a certainamount of time, and generating B-cell hybridomas from the subject thatexpress a monoclonal antibody that binds to and neutralizes strains oftwo or more Influenza A virus HA subtypes, wherein each immunogeniccomposition comprises an inactivated Influenza virus, an attenuatedInfluenza virus, a live Influenza virus other than an attenuatedInfluenza virus (e.g., naturally occurring Influenza virus), an antigenderived or obtained from an Influenza virus, or a nucleic acid encodingan antigen derived or obtained from an Influenza virus, and wherein oneimmunogenic composition differs from another immunogenic composition inthat the Influenza virus, or the Influenza virus from which the antigenor the nucleic acid encoding the antigen is derived or obtained areantigenically distinct. In some embodiments, the method comprisesselecting hybridoma clones that express a monoclonal antibody that bindsto and neutralizes strains of Influenza A virus of two or more HAsubtypes. In certain embodiments, the method comprises selectinghybridoma clones that express a monoclonal antibody that binds to andneutralizes two or more strains of an Influenza A virus subtype.

In one embodiment, a method for generating a monoclonal antibody thatbinds to and neutralizes strains of Influenza A virus of two or more HAsubtypes comprises: (i) administering to a non-human subject a firstimmunogenic composition comprising an inactivated first Influenza virus,an attenuated first Influenza virus, a live first Influenza virus otherthan an attenuated Influenza virus, an HA derived or obtained from afirst Influenza virus or fragment thereof, or a nucleic acid encoding anHA derived or obtained from a first Influenza virus or fragment thereof;(ii) after a first period of time, administering to the subject a secondimmunogenic composition comprising an inactivated second Influenzavirus, an attenuated second Influenza virus, a live second Influenzavirus other than an attenuated Influenza virus, an HA derived orobtained from a second Influenza virus or fragment thereof, or a nucleicacid encoding an HA derived or obtained from a second Influenza virus orfragment thereof, wherein the second Influenza virus is antigenicallydistinct from the first Influenza virus; and (iii) after a second periodof time, generating B-cell hybridomas from the subject that express amonoclonal antibody that binds to and neutralizes strains of Influenza Avirus of two or more HA subtypes. In some embodiments, the methodcomprises selecting hybridoma clones that express a monoclonal antibodythat binds to and neutralizes strains of Influenza A virus of two ormore HA subtypes. In certain embodiments, the method comprises selectinghybridoma clones that express a monoclonal antibody that binds to andneutralizes two or more strains of an Influenza A virus subtype. Incertain embodiments, the method further comprises isolating themonoclonal antibody. In some embodiments, the method further comprisesscreening for the cross-reactive neutralizing monoclonal antibody. Inspecific embodiments, the Influenza A virus subtypes are H1 and H3.

In another embodiment, a method for generating a monoclonal antibodythat binds to and neutralizes strains of Influenza A virus of two ormore HA subtypes comprises: (i) administering to a non-human subject afirst immunogenic composition comprising an inactivated first Influenzavirus, an attenuated first Influenza virus, a live first Influenza virusother than an attenuated Influenza virus, an HA derived or obtained froma first Influenza virus or fragment thereof, or a nucleic acid encodingan HA derived or obtained from a first Influenza virus or fragmentthereof; (ii) after a first period of time, administering to the subjecta second immunogenic composition comprising an inactivated secondInfluenza virus, an attenuated second Influenza virus, a live secondInfluenza virus other than an attenuated Influenza virus, an HA derivedor obtained from a second Influenza virus or fragment thereof, or anucleic acid encoding an HA derived or obtained from a second Influenzavirus or fragment thereof, wherein the second Influenza virus isantigenically distinct from the first Influenza virus; (iii) after asecond period of time, administering to the subject a third immunogeniccomposition comprising an inactivated third Influenza virus, anattenuated third Influenza virus, a live third Influenza virus otherthan an attenuated Influenza virus, an HA derived or obtained from athird Influenza virus or fragment thereof, or a nucleic acid encoding anHA derived or obtained from a third Influenza virus or fragment thereof,wherein the third Influenza virus is antigenically distinct from thefirst and the second Influenza viruses; and (iv) after a third period oftime, generating B-cell hybridomas from the subject that express amonoclonal antibody that binds to and neutralizes strains of Influenza Avirus of two or more HA subtypes. In some embodiments, the methodcomprises selecting hybridoma clones that express a monoclonal antibodythat binds to and neutralizes strains of Influenza A virus of two ormore HA subtypes. In certain embodiments, the method comprises selectinghybridoma clones that express a monoclonal antibody that binds to andneutralizes two or more strains of an Influenza A virus subtype. Incertain embodiments, the method further comprises isolating themonoclonal antibody. In some embodiments, the method further comprisesscreening for the cross-reactive neutralizing monoclonal antibody. Inspecific embodiments, the Influenza A virus subtypes are H1 and H3.

In another embodiment, a method for generating a monoclonal antibodythat binds to and neutralizes strains of Influenza A virus of two ormore HA subtypes comprises: (i) administering to a non-human subject afirst immunogenic composition comprising an inactivated first Influenzavirus, an attenuated first Influenza virus, a live first Influenza virusother than an attenuated Influenza virus, an HA derived or obtained froma first Influenza virus or fragment thereof, or a nucleic acid encodingan HA derived or obtained from a first Influenza virus or fragmentthereof; (ii) after a first period of time, administering to the subjecta second immunogenic composition comprising an inactivated secondInfluenza virus, an attenuated second Influenza virus, a live secondInfluenza virus other than an attenuated Influenza virus, an HA derivedor obtained from a second Influenza virus or fragment thereof, or anucleic acid encoding an HA derived or obtained from a second Influenzavirus or fragment thereof, wherein the second Influenza virus isantigenically distinct from the first Influenza virus; (iii) after asecond period of time, administering to the subject a third immunogeniccomposition comprising an inactivated third Influenza virus, anattenuated third Influenza virus, a live third Influenza virus otherthan an attenuated Influenza virus, an HA derived or obtained from athird Influenza virus or fragment thereof, or a nucleic acid encoding anHA derived or obtained from a third Influenza virus or fragment thereof,wherein the third Influenza virus is antigenically distinct from thefirst and the second Influenza viruses; (iv) after a third period oftime, administering to the subject a fourth immunogenic compositioncomprising an inactivated fourth Influenza virus, an attenuated fourthInfluenza virus, a live fourth Influenza virus other than an attenuatedInfluenza virus, an HA derived or obtained from a fourth Influenza virusor fragment thereof, or a nucleic acid encoding an HA derived orobtained from a fourth Influenza virus or fragment thereof, wherein thefourth Influenza virus is antigenically distinct from the first, secondand third Influenza viruses; and (v) after a fourth period of time,generating B-cell hybridomas from the subject and further selecting forhybridoma clones that express a monoclonal antibody that binds to andneutralizes strains of Influenza A virus of two or more HA subtypes. Insome embodiments, the method comprises selecting hybridoma clones thatexpress a monoclonal antibody that binds to and neutralizes strains ofInfluenza A virus of two or more HA subtypes. In certain embodiments,the method comprises selecting hybridoma clones that express amonoclonal antibody that binds to and neutralizes two or more strains ofan Influenza A virus subtype. In certain embodiments, the method furthercomprises isolating the monoclonal antibody. In some embodiments, themethod further comprises screening for the cross-reactive neutralizingmonoclonal antibody. In specific embodiments, the Influenza A virussubtypes are H1 and H3.

In another embodiment, a method for generating a monoclonal antibodythat binds to and neutralizes strains of Influenza A virus of two ormore HA subtypes comprises: (i) administering to a non-human subject afirst immunogenic composition comprising an inactivated first Influenzavirus of a first HA subtype, an attenuated first Influenza virus of afirst HA subtype, a live first Influenza virus of a first HA subtypeother than an attenuated Influenza virus, an HA of a first HA subtypederived or obtained from a first Influenza virus of a first HA subtypeof a first HA subtype or fragment thereof, or a nucleic acid encoding anHA of a first HA subtype derived or obtained from a first Influenzavirus or fragment thereof; (ii) after a first period of time,administering to the subject a second immunogenic composition comprisingan inactivated second Influenza virus of a second HA subtype, anattenuated second Influenza virus of a second HA subtype, a live secondInfluenza virus of a second HA subtype other than an attenuatedInfluenza virus, an HA of a second HA subtype derived or obtained from asecond Influenza virus or fragment thereof, or a nucleic acid encodingan HA of a second HA subtype derived or obtained from a second Influenzavirus or fragment thereof, wherein the second Influenza virus isantigenically distinct from the first Influenza virus; (iii) after asecond period of time, administering to the subject a third immunogeniccomposition comprising an inactivated third Influenza virus of a thirdHA subtype, an attenuated third Influenza virus of a third HA subtype, alive third Influenza virus of a third HA subtype other than anattenuated Influenza virus, an HA of a third HA subtype derived orobtained from a third Influenza virus or fragment thereof, or a nucleicacid encoding an HA of a third HA subtype derived or obtained from athird Influenza virus or fragment thereof, wherein the third Influenzavirus is antigenically distinct from the first and the second Influenzaviruses; (iv) after a third period of time, administering to the subjecta fourth immunogenic composition comprising an inactivated fourthInfluenza virus of a fourth HA subtype, an attenuated fourth Influenzavirus of a fourth HA subtype, a live fourth Influenza virus of a fourthHA subtype other than an attenuated Influenza virus, an HA of a fourthHA subtype derived or obtained from a fourth Influenza virus or fragmentthereof, or a nucleic acid encoding an HA of a fourth HA subtype derivedor obtained from a fourth Influenza virus or fragment thereof, whereinthe fourth Influenza virus is antigenically distinct from the first,second and third Influenza viruses; (v) after a fourth period of time,administering to the subject a fifth immunogenic composition comprising(a) an inactivated fifth Influenza virus of a fifth HA subtype, anattenuated fifth Influenza virus of a fifth HA subtype, a live fifthInfluenza virus of a fifth HA subtype other than an attenuated Influenzavirus, an HA of a fifth HA subtype derived or obtained from a fifthInfluenza virus or fragment thereof, or a nucleic acid encoding an HA ofa fifth HA subtype derived or obtained from a fifth Influenza virus orfragment thereof and (b) an inactivated sixth Influenza virus of a sixthHA subtype, an attenuated sixth Influenza virus of a sixth HA subtype, alive sixth Influenza virus of a sixth HA subtype other than anattenuated Influenza virus, an HA of a sixth HA subtype derived orobtained from a sixth Influenza virus or fragment thereof, or a nucleicacid encoding an HA of a sixth HA subtype derived or obtained from asixth Influenza virus or fragment thereof, wherein the fifth Influenzavirus is antigenically distinct from the first, second, third, andfourth Influenza viruses and wherein the sixth Influenza virus isantigenically distinct from the first, second, third, fourth, and fifthInfluenza viruses; and (vi) after a fifth period of time, generatingB-cell hybridomas from the subject and further selecting for hybridomaclones that express a monoclonal antibody that binds to and neutralizesstrains of Influenza A virus of two or more HA subtypes. In certainembodiments, the first, third, and fifth HA subtypes are the same (e.g.,the subtypes are all H3 subtypes) and the second, fourth, and sixth HAsubtypes are the same (e.g., the subtypes are all H1 subtypes). Inspecific embodiments, the Influenza A virus subtypes are H1 and H3. Inspecific embodiments, the first Influenza virus is A/Hong Kong/1/68(H3), the second Influenza virus is A/USSR/92/77 (H1), the thirdInfluenza virus is A/California/1/88 (H3), the fourth Influenza virus isA/California/04/09 (H1), the fifth Influenza virus isA/Brisbane/59/07-like (H1), and the sixth Influenza virus isA/Brisbane/10/07-like (H3). In some embodiments, the first, second,third, fourth, fifth, and sixth HA subtypes are 2, 3, or more differentsubtypes. In some embodiments, the method comprises selecting hybridomaclones that express a monoclonal antibody that binds to and neutralizesstrains of Influenza A virus of two or more HA subtypes. In certainembodiments, the method comprises selecting hybridoma clones thatexpress a monoclonal antibody that binds to and neutralizes two or morestrains of an Influenza A virus subtype. In certain embodiments, themethod further comprises isolating the monoclonal antibody. In someembodiments, the method further comprises screening for thecross-reactive neutralizing monoclonal antibody.

In certain embodiments, the non-human subject referenced in the methodsdescribed herein is a transgenic animal (e.g., a transgenic mouse)capable of producing human antibodies. Examples of transgenic mice thatare capable of producing human antibodies are those available fromAbgenix, Inc. (Freemont, Calif.), Genpharm (San Jose, Calif.), orMedarex, Inc. (Princeton, N.J.).

In another aspect, provided herein are isolated antibodies (e.g.,monoclonal antibodies and antigen-binding fragments thereof) that bindto and neutralize two or more strains of an Influenza A virus. In aspecific embodiment, the strains are from the same Influenza A virus HAsubtype. In another specific embodiment, the strains are Influenza Aviruses belonging to different HA subtypes. In certain embodiments, suchmonoclonal antibodies are humanized.

In a specific embodiment, provided herein are the antibody 7A7, 12D1,39A4, 66A6 or a fragment thereof (in particular, an antigen-bindingfragment thereof). In another specific embodiment, provided herein is anantibody that binds to Influenza virus HA, wherein the antibodycomprises the variable heavy (VH) domain of the antibody 7A7, 12D1,39A4, or 66A6. In another specific embodiment, provided herein is anantibody that binds to Influenza virus HA, wherein the antibodycomprises the variable light (VL) domain of the antibody 7A7, 12D1,39A4, or 66A6. In another specific embodiment, provided herein is anantibody that binds to Influenza virus HA, wherein the antibodycomprises the VH and VL domain of the antibody 7A7, 12D1, 39A4, or 66A6.In another specific embodiment, provided herein is an antibody thatbinds to Influenza virus HA, wherein the antibody comprises 1, 2, or 3VH CDRs and/or 1, 2, or 3 VL CDRs of the antibody 7A7, 12D1, 39A4, or66A6. In certain embodiments, the antibody not only binds to Influenzavirus HA, but also neutralizes the Influenza virus.

Also provided herein are nucleic acids encoding the antibodies providedherein or generated in accordance with the methods provided herein. In aspecific embodiment, a nucleic acid(s) provided herein encodes for theantibody 7A7, 12D1, 39A4, 66A6 or a fragment thereof (in particular, anantigen-binding fragment thereof). In another specific embodiment, anucleic acid(s) provided herein encodes for an antibody that binds toInfluenza virus HA, wherein the antibody comprises the VH domain of theantibody 7A7, 12D1, 39A4, or 66A6. In another specific embodiment, anucleic acid(s) provided herein encodes for an antibody that binds toInfluenza virus HA, wherein the antibody comprises the VL domain of theantibody 7A7, 12D1, 39A4, or 66A6. In another specific embodiment, anucleic acid(s) provided herein encodes for an antibody that binds toInfluenza virus HA, wherein the antibody comprises the VH and VL domainof the antibody 7A7, 12D1, 39A4, or 66A6. In another specificembodiment, a nucleic acid(s) provided herein encodes for an antibodythat binds to Influenza virus HA, wherein the antibody comprises 1, 2,or 3 VH CDRs and/or 1, 2, or 3 VL CDRs of the antibody 7A7, 12D1, 39A4,or 66A6. In certain embodiments, the nucleic acid encodes an antibodythat not only binds to Influenza virus HA, but also neutralizes theInfluenza virus.

In another aspect, provided herein are hybridomas produced in accordancewith the methods described herein. In one embodiment, provided herein isa hybridoma designated 7A7 deposited under provisions of the BudapestTreaty with the American Type Culture Collection (ATCC, 10801 UniversityBlvd., Manassas, Va. 20110-2209) on May 22, 2009 (ATCC Accession No.PTA-10058). In another embodiment, provided herein is a hybridomadesignated 12D1 deposited under provisions of the Budapest Treaty withthe American Type Culture Collection (ATCC, 10801 University Blvd.,Manassas, Va. 20110-2209) on May 22, 2009 (ATCC Accession No.PTA-10059). In another embodiment, provided herein is a hybridomadesignated 39A4 deposited under provisions of the Budapest Treaty withthe American Type Culture Collection (ATCC, 10801 University Blvd.,Manassas, Va. 20110-2209) on May 22, 2009 (ATCC Accession No.PTA-10060). In another embodiment, provided herein is a hybridomadesignated 66A6 deposited under provisions of the Budapest Treaty withthe American Type Culture Collection (ATCC, 10801 University Blvd.,Manassas, Va. 20110-2209) on May 25, 2010 (ATCC Accession No.PTA-______).

In another aspect, provided herein are isolated monoclonal antibodies orantigen-binding fragments thereof produced by the hybridomas generatedin the accordance with the methods described herein. In a specificembodiment, the isolated monoclonal antibody is the antibody 7A7, 12D1or 39A4. In certain embodiments, such monoclonal antibodies arehumanized.

In another aspect, provided herein are isolated antibodies that bind toSEQ ID NO:1 and neutralize two or more strains of an Influenza A virusHA subtype. In another aspect, provided herein are compositions as wellas kits comprising an antibody described herein. In a specificembodiment, such compositions are pharmaceutical compositions suitablefor administration to a patient.

In another aspect, provided herein are methods of preventing and/ortreating an Influenza virus disease comprising administering to asubject an antibody described herein or a pharmaceutical compositionthereof. In a specific embodiment, provided herein are methods forpreventing and/or treating Influenza virus infection comprisingadministering to a subject an antibody described herein or apharmaceutical composition thereof.

In another aspect, provided herein are methods for detecting anInfluenza virus, or detecting, diagnosing or monitoring an Influenzavirus infection in a subject using an antibody described herein. In aspecific embodiment, a method of detecting a strain of Influenza A viruscomprises: (a) assaying for the level of an Influenza virus HA in cellsor a tissue sample of a subject using an antibody; and (b) comparing thelevel of the Influenza virus HA assayed in (a) with the level of theInfluenza virus HA in cells or tissue samples not infected withInfluenza virus (e.g., a control level), wherein an increase in theassayed level of Influenza virus HA compared to the control level of theInfluenza virus antigen is indicative of the presence of a strain ofInfluenza A virus. In specific embodiments, the strain of Influenza Avirus detected belongs to the H3 subtype. In a specific embodiment, thestrain of Influenza A virus detected is Influenza virus is A/HongKong/1/1968, A/Alabama/1/1981, A/Beijing/47/1992, or A/Wyoming/3/2003.

3.1 Terminology

As used herein, the term “about” or “approximately” when used inconjunction with a number refers to any number within 0.25%, 0.5%, 1%,5% or 10% of the referenced number. The terms “about” or “approximate,”when used in reference to an amino acid position refer to the particularamino acid position in a sequence or any amino acid that is within five,four, three, two or one residues of that amino acid position, either inan N-terminal direction or a C-terminal direction.

As used herein, the term “administer” or “administration” refers to theact of injecting or otherwise physically delivering a substance as itexists outside the body (e.g., an anti-Influenza A virus antibodyprovided herein) into a patient, such as by mucosal, intradermal,intravenous, intramuscular delivery and/or any other method of physicaldelivery described herein or known in the art. When a disease, or asymptom thereof, is being treated, administration of the substancetypically occurs after the onset of the disease or symptoms thereof.When a disease, or symptoms thereof, are being prevented, administrationof the substance typically occurs before the onset of the disease orsymptoms thereof.

Antibodies encompassed herein include, but are not limited to, syntheticantibodies, monoclonal antibodies, recombinantly produced antibodies,multispecific antibodies (including bispecific antibodies), humanantibodies, humanized antibodies, chimeric antibodies, intrabodies,single-chain Fvs (scFv) (e.g., including monospecific, bispecific,etc.), camelized antibodies, Fab fragments, F(ab′) fragments, F(ab′)₂fragments, disulfide-linked Fvs (sdFv), anti-idiotypic (anti-Id)antibodies, and epitope-binding fragments of any of the above. Inparticular, antibodies encompassed herein include immunoglobulinmolecules and immunologically active portions of immunoglobulinmolecules, i.e., antigen binding domains or molecules that contain anantigen-binding site that immunospecifically binds to an Influenza virusantigen (e.g., one or more complementarity determining regions (CDRs) ofan anti-Influenza virus antibody). The antibodies encompassed herein canbe of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), any class (e.g.,IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2), or any subclass (e.g., IgG2a andIgG2b) of immunoglobulin molecule.

As used herein, the term “effective amount” in the context ofadministering a therapy to a subject refers to the amount of a therapywhich has a prophylactic and/or therapeutic effect(s). In certainembodiments, an “effective amount” in the context of administration of atherapy to a subject refers to the amount of a therapy which issufficient to achieve one, two, three, four, or more of the followingeffects: (i) reduction or amelioration the severity of an Influenzavirus infection, an Influenza virus disease or a symptom associatedtherewith; (ii) reduction in the duration of an Influenza virusinfection, an Influenza virus disease or a symptom associated therewith;(iii) prevention of the progression of an Influenza virus infection, anInfluenza virus disease or a symptom associated therewith; (iv)regression of an Influenza virus infection, an Influenza virus diseaseor a symptom associated therewith; (v) prevention of the development oronset of an Influenza virus infection, an Influenza virus disease or asymptom associated therewith; (vi) prevention of the recurrence of anInfluenza virus infection, an Influenza virus disease or a symptomassociated therewith; (vii) reduction or prevention of the spread of anInfluenza virus from one cell to another cell, one tissue to anothertissue, or one organ to another organ; (viii) prevention or reduction ofthe spread/transmission of an Influenza virus from one subject toanother subject; (ix) reduction in organ failure associated with anInfluenza virus infection or Influenza virus disease; (x) reduction inthe hospitalization of a subject; (xi) reduction in the hospitalizationlength; (xii) an increase in the survival of a subject with an Influenzavirus infection or a disease associated therewith; (xiii) elimination ofan Influenza virus infection or a disease associated therewith; (xiv)inhibition or reduction in Influenza virus replication; (xv) inhibitionor reduction in the binding or fusion of Influenza virus to a hostcell(s); (xvi) inhibition or reduction in the entry of an Influenzavirus into a host cell(s); (xvii) inhibition or reduction of replicationof the Influenza virus genome; (xviii) inhibition or reduction in thesynthesis of Influenza virus proteins; (xix) inhibition or reduction inthe assembly of Influenza virus particles; (xx) inhibition or reductionin the release of Influenza virus particles from a host cell(s); (xxi)reduction in Influenza virus titer; (xxii) the reduction in the numberof symptoms associated with an Influenza virus infection or an Influenzavirus disease; (xxiii) enhancement, improvement, supplementation,complementation, or augmentation of the prophylactic or therapeuticeffect(s) of another therapy; (xxiv) prevention of the onset orprogression of a secondary infection associated with an Influenza virusinfection; and/or (xxv) prevention of the onset or diminution of diseaseseverity of bacterial pneumonias occurring secondary to Influenza virusinfections. In some embodiments, the “effective amount” of a therapy hasa beneficial effect but does not cure an Influenza virus infection or adisease associated therewith. In certain embodiments, the “effectiveamount” of a therapy may encompass the administration of multiple dosesof a therapy at a certain frequency to achieve an amount of the therapythat has a prophylactic and/or therapeutic effect. In other situations,the “effective amount” of a therapy may encompass the administration ofa single dose of a therapy at a certain amount. Exemplary doses of aneffective amount are provided in Section 5.5.2 infra.

In certain embodiments, the effective amount does not result in completeprotection from an Influenza virus disease, but results in a lower titeror reduced number of Influenza viruses compared to an untreated subject.In certain embodiments, the effective amount results in a 0.5 fold, 1fold, 2 fold, 4 fold, 6 fold, 8 fold, 10 fold, 15 fold, 20 fold, 25fold, 50 fold, 75 fold, 100 fold, 125 fold, 150 fold, 175 fold, 200fold, 300 fold, 400 fold, 500 fold, 750 fold, or 1,000 fold or greaterreduction in titer of Influenza virus relative to an untreated subject.In some embodiments, the effective amount results in a reduction intiter of Influenza virus relative to an untreated subject ofapproximately 1 log or more, approximately 2 logs or more, approximately3 logs or more, approximately 4 logs or more, approximately 5 logs ormore, approximately 6 logs or more, approximately 7 logs or more,approximately 8 logs or more, approximately 9 logs or more,approximately 10 logs or more, 1 to 5 logs, 2 to 10 logs, 2 to 5 logs,or 2 to 10 logs. Benefits of a reduction in the titer, number or totalburden of Influenza virus include, but are not limited to, less severesymptoms of the infection, fewer symptoms of the infection, reduction inthe length of the disease associated with the infection, and preventionof the onset or diminution of disease severity of bacterial pneumoniasoccurring secondary to Influenza virus infections.

As used herein, the term “fragment” refers to a sequence comprising atleast 2 consecutive amino acids or nucleotides from a parent sequence.In a specific embodiment, it refers to 2 to 10, 2 to 15, 2 to 30, 5 to30, 10 to 60, 25 to 50, 25 to 100, 100 to 175, 150 to 250, 150 to 300 ormore consecutive amino acids or nucleotides from a parent sequence.

As used herein, the terms “hemagglutinin” and “HA” refer to anyInfluenza hemagglutinin known to those of skill in the art. In certainembodiments, the hemagglutinin is Influenza hemagglutinin, such as anInfluenza A hemagglutinin, an Influenza B hemagglutinin or an InfluenzaC hemagglutinin. A typical hemagglutinin comprises domains known tothose of skill in the art including a signal peptide, a stem domain, aglobular head domain, a luminal domain, a transmembrane domain and acytoplasmic domain. In certain embodiments, a hemagglutinin consists ofa single polypeptide chain, such as HA0. In certain embodiments, ahemagglutinin consists of more than one polypeptide chain in quaternaryassociation, e.g. HA1 and HA2. In certain embodiments, a hemagglutininconsists of an hemagglutinin monomer (HA0 or HA1/HA2). In certainembodiments, a hemagglutinin consists of a trimeric hemagglutininmolecule as it is expressed on the viral surface. Those of skill in theart will recognize that an immature HA0 might be cleaved to release asignal peptide (approximately 20 amino acids) yielding a maturehemagglutinin HA0. A hemagglutinin HA0 might be cleaved at another siteto yield HA1 polypeptide (approximately 320 amino acids, including theglobular head domain and a portion of the stem domain) and HA2polypeptide (approximately 220 amino acids, including the remainder ofthe stem domain, a luminal domain, a transmembrane domain and acytoplasmic domain). In certain embodiments, a hemagglutinin comprises asignal peptide, a transmembrane domain and a cytoplasmic domain. Incertain embodiments, a hemagglutinin lacks a signal peptide, i.e. thehemagglutinin is a mature hemagglutinin. In certain embodiments, ahemagglutinin lacks a transmembrane domain or cytoplasmic domain, orboth. As used herein, the terms “hemagglutinin” and “HA” encompasshemagglutinin polypeptides that are modified by post-translationalprocessing such as signal peptide cleavage, disulfide bond formation,glycosylation (e.g., N-linked glycosylation), protease cleavage andlipid modification (e.g. S-palmitoylation).

As used herein, the term “host cell” refers to any type of cell, e.g., aprimary cell or a cell from a cell line. In specific embodiments, theterm “host cell” refers a cell transfected with a nucleic acid moleculeand the progeny or potential progeny of such a cell. Progeny of such acell may not be identical to the parent cell transfected with thenucleic acid molecule due to mutations or environmental influences thatmay occur in succeeding generations or integration of the nucleic acidmolecule into the host cell genome.

As used herein, the term “infection” means the invasion by,multiplication and/or presence of a virus in a cell or a subject. In oneembodiment, an infection is an “active” infection, i.e., one in whichthe virus is replicating in a cell or a subject. Such an infection ischaracterized by the spread of the virus to other cells, tissues, and/ororgans, from the cells, tissues, and/or organs initially infected by thevirus. An infection may also be a latent infection, i.e., one in whichthe virus is not replicating. In certain embodiments, an infectionrefers to the pathological state resulting from the presence of thevirus in a cell or a subject, or by the invasion of a cell or subject bythe virus. In certain embodiments, an infection refers to the presenceof a virus in a cell or a subject, or the invasion of a cell or subjectby the virus, without a resulting pathological state.

As used herein, the term “Influenza virus antigen” refers to any antigenobtained or derived from an Influenza virus. In a specific embodiment,the antigen is a protein, polypeptide or peptide expressed by anInfluenza virus or a fragment thereof. In another specific embodiment,the antigen is a protein, polypeptide or peptide found on the surface ofan Influenza virus particle or a fragment thereof (e.g., hemagglutininor a fragment thereof, or neuraminidase or a fragment thereof). In apreferred embodiment, the antigen is an Influenza virus hemagglutinin ora fragment thereof.

As used herein, the terms “Influenza virus disease” and a diseaseassociated with an Influenza virus infection refer to the pathologicalstate resulting from the presence of an Influenza virus (e.g., InfluenzaA or B virus) in a cell or subject or the invasion of a cell or subjectby an Influenza virus. In specific embodiments, the term refers to arespiratory illness caused by an Influenza virus.

The terms “Kabat numbering,” and like terms are recognized in the artand refer to a system of numbering amino acid residues which are morevariable (i.e. hypervariable) than other amino acid residues in theheavy and light chain variable regions of an antibody, or an antigenbinding portion thereof (Kabat et al. (1971) Ann. NY Acad. Sci.190:382-391 and, Kabat et al. (1991) Sequences of Proteins ofImmunological Interest, Fifth Edition, U.S. Department of Health andHuman Services, NIH Publication No. 91-3242). For the heavy chainvariable region, the hypervariable region typically ranges from aminoacid positions 31 to 35 for CDR1, amino acid positions 50 to 65 forCDR2, and amino acid positions 95 to 102 for CDR3. For the light chainvariable region, the hypervariable region typically ranges from aminoacid positions 24 to 34 for CDR1, amino acid positions 50 to 56 forCDR2, and amino acid positions 89 to 97 for CDR3.

An “isolated” or “purified” protein (e.g., an antibody) is substantiallyfree of cellular material or other contaminating proteins from the cellor tissue source from which the protein is derived, or substantiallyfree of chemical precursors or other chemicals when chemicallysynthesized. The language “substantially free of cellular material”includes preparations of a protein (e.g., an antibody) in which theprotein is separated from cellular components of the cells from which itis isolated or recombinantly produced. Thus, a protein (e.g., anantibody) that is substantially free of cellular material includespreparations of protein having less than about 30%, 20%, 10%, or 5% (bydry weight) of heterologous protein (also referred to herein as a“contaminating protein”). When the protein is recombinantly produced, itis also preferably substantially free of culture medium, i.e., culturemedium represents less than about 20%, 10%, or 5% of the volume of theprotein preparation. When the protein is produced by chemical synthesis,it is preferably substantially free of chemical precursors or otherchemicals, i.e., it is separated from chemical precursors or otherchemicals which are involved in the synthesis of the protein.Accordingly such preparations of the protein have less than about 30%,20%, 10%, 5% (by dry weight) of chemical precursors or compounds otherthan the protein of interest. In a specific embodiment, an antigenderived or obtained from an Influenza virus is purified. In anotherspecific embodiment, antibodies encompassed herein are purified.

As used herein, the numeric term “log” refers to log₁₀.

As used herein, the terms “manage,” “managing,” and “management” referto the beneficial effects that a subject derives from a therapy (e.g., aprophylactic or therapeutic agent), which does not result in a cure ofthe infection or disease associated therewith. In certain embodiments, asubject is administered one or more therapies (e.g., prophylactic ortherapeutic agents, such as an antibody encompassed herein) to “manage”an Influenza virus disease, or one or more symptoms thereof, so as toprevent the progression or worsening of the disease.

As used herein, the terms “nucleic acid” and “nucleotides” refer todeoxyribonucleotides, deoxyribonucleic acids, ribonucleotides, andribonucleic acids, and polymeric forms thereof, and includes eithersingle- or double-stranded forms. In certain embodiments, such termsinclude known analogues of natural nucleotides, for example, peptidenucleic acids (“PNA”s), that have similar binding properties as thereference nucleic acid. In some embodiments, nucleic acid refers todeoxyribonucleic acids (e.g., cDNA or DNA). In other embodiments,nucleic acid refers to ribonucleic acid (e.g., mRNA or RNA).

As used herein, the terms “prevent,” “preventing” and “prevention” inthe context of the administration of a therapy(ies) to a subject toprevent an Influenza virus disease refer to one or more of the followingeffects resulting from the administration of a therapy or a combinationof therapies: (i) the inhibition or reduction in the development oronset of an Influenza virus disease or a symptom thereof (e.g., fever,myalgia, edema, inflammatory infiltrates); (ii) the inhibition orreduction in the recurrence of an Influenza virus disease or a symptomassociated therewith; and (iii) the reduction or inhibition in Influenzavirus infection and/or replication.

As used herein, the terms “prevent”, “preventing” and “prevention” inthe context of the administration of a therapy(ies) to a subject toprevent an Influenza virus infection refer to one or more of thefollowing: (i) the reduction or inhibition of the spread of Influenzavirus from one cell to another cell; (ii) the reduction or inhibition ofthe spread of Influenza virus from one organ or tissue to another organor tissue; (iii) the reduction or inhibition of the spread of Influenzavirus from one region of an organ or tissue to another region of theorgan or tissue (e.g., the reduction in the spread of Influenza virusfrom the upper to lower respiratory tract); (iv) the prevention of aninitial infection after exposure to an Influenza virus; and/or (v)prevention of the onset or development of one or more symptomsassociated with Influenza virus disease or infection.

As used herein, the terms “subject” and “patient” are usedinterchangeably to refer to an animal (e.g., birds, reptiles, andmammals). In a specific embodiment, a subject is a bird. In anotherembodiment, a subject is a mammal including a non-primate (e.g., acamel, donkey, zebra, cow, pig, horse, goat, sheep, cat, dog, rat, andmouse) and a primate (e.g., a monkey, chimpanzee, and a human). Inanother embodiment, a subject is a human. In another embodiment, asubject is a human infant. In another embodiment, a subject is a humanchild. In another embodiment, the subject is a human adult. In anotherembodiment, a subject is an elderly human.

As used herein, the term “human infant” refers to a newborn to 1 yearold human.

As used herein, the term “human child” refers to a human that is 1 yearto 18 years old.

As used herein, the term “human toddler” refers to a human that is 1years to 3 years old.

As used herein, the term “human adult” refers to a human that is 18years or older.

As used herein, the term “elderly human” refers to a human 65 years orolder.

As used herein, the terms “therapies” and “therapy” can refer to anyprotocol(s), method(s), compound(s), composition(s), formulation(s),and/or agent(s) that can be used in the prevention or treatment of aviral infection or a disease or symptom associated therewith. In certainembodiments, the terms “therapies” and “therapy” refer to biologicaltherapy, supportive therapy, and/or other therapies useful in treatmentor prevention of a viral infection or a disease or symptom associatedtherewith known to one of skill in the art. In some embodiments, theterm “therapy” refers to an antibody that binds an Influenza virus ofthe hemagglutinin 3 (“H3”) subtype. In other embodiments, the term“therapy” refers to an immunogenic composition (e.g., an Influenza virusvaccine).

As used herein, the terms “treat,” “treatment,” and “treating” in thecontext of administration of a therapy(ies) to a subject to treat anInfluenza virus disease or Influenza virus infection refer to abeneficial or therapeutic effect of a therapy or a combination oftherapies. In specific embodiments, such terms refer to one, two, three,four, five or more of the following effects resulting from theadministration of a therapy or a combination of therapies: (i) reductionor amelioration in the severity of an Influenza virus infection, anInfluenza virus disease or a symptom associated therewith; (ii)reduction in the duration of an Influenza virus infection, an Influenzavirus disease or a symptom associated therewith; (iii) prevention of theprogression of an Influenza virus infection, an Influenza virus diseaseor a symptom associated therewith; (iv) regression of an Influenza virusinfection, an Influenza virus disease or a symptom associated therewith;(v) prevention of the development or onset of an Influenza virusinfection, an Influenza virus disease or a symptom associated therewith;(vi) prevention of the recurrence of an Influenza virus infection, anInfluenza virus disease or a symptom associated therewith; (vii)reduction or prevention of the spread of an Influenza virus from onecell to another cell, one tissue to another tissue, or one organ toanother organ; (viii) prevention or reduction of the spread/transmissionof an Influenza virus from one subject to another subject; (ix)reduction in organ failure associated with an Influenza virus infectionor Influenza virus disease; (x) reduction in the hospitalization of asubject; (xi) reduction in the hospitalization length; (xii) an increasein the survival of a subject with an Influenza virus infection or adisease associated therewith; (xiii) elimination of an Influenza virusinfection or a disease associated therewith; (xiv) inhibition orreduction in Influenza virus replication; (xv) inhibition or reductionin the binding or fusion of Influenza virus to a host cell(s); (xvi)inhibition or reduction in the entry of an Influenza virus into a hostcell(s); (xvii) inhibition or reduction of replication of the Influenzavirus genome; (xviii) inhibition or reduction in the synthesis ofInfluenza virus proteins; (xix) inhibition or reduction in the assemblyof Influenza virus particles; (xx) inhibition or reduction in therelease of Influenza virus particles from a host cell(s); (xxi)reduction in Influenza virus titer; (xxii) the reduction in the numberof symptoms associated with an Influenza virus infection or an Influenzavirus disease; (xxiii) enhancement, improvement, supplementation,complementation, or augmentation of the prophylactic or therapeuticeffect(s) of another therapy; (xxiv) prevention of the onset orprogression of a secondary infection associated with an Influenza virusinfection; and/or (xxv) prevention of the onset or diminution of diseaseseverity of bacterial pneumonias occurring secondary to Influenza virusinfections.

4 DESCRIPTION OF THE FIGURES

FIG. 1. Characteristics of anti-Influenza virus antibodies. (A)Monoclonal antibodies 7A7 and 39A4 react with Influenza virusA/HK/1/1968 hemagglutinin as measured by ELISA; and monoclonal antibody12D1 reacts with A/HK/1/1968 hemagglutinin as measured by Western blot.(B) Monoclonal antibody 12D1 binds hemagglutinin of Influenza virusstrain A/Pan/2007/1999 (H3) in the HA2 region as measured by WesternBlot. (C) Monoclonal antibody 7A7 binds Influenza virus strainsA/HK/1/1968, A/Pan/2007/1999, and A/Wisc/67/2005 as measured by ELISA.(D) Monoclonal antibody 39A4 binds Influenza virus strains A/HK/1/1968,A/Pan/2007/1999, and A/Wisc/67/2005 as measured by ELISA.

FIG. 2. Neutralization of Influenza virus strains by monoclonalantibodies 7A7, 12D1, and 39A4. (A) Monoclonal antibodies 7A7, 12D1, and39A4 neutralize Influenza virus strain A/HK/1/1968 (H3) as measured bymicroneutralization assay. IgG represents isotype control antibody;XY102 represents a monoclonal antibody specific to Influenza virusstrain A/HK/1/1968 (H3). (B) Monoclonal antibodies 7A7, 12D1, and 39A4neutralize Influenza virus strain A/Pan/2007/1999 (H3) as measured bymicroneutralization assay. IgG represents isotype control antibody;XY102 represents a monoclonal antibody specific to Influenza virusstrain A/HK/1/1968 (H3).

FIG. 3. Monoclonal antibodies 7A7, 12D1, and 39A4 specificallyneutralize Influenza virus H3 strains. (A) Neutralization by monoclonalantibody 7A7 as measured by plaque reduction assay. (B) Neutralizationby monoclonal antibody 12D1 as measured by plaque reduction assay. (C)Neutralization by monoclonal antibody 39A4 as measured by plaquereduction assay. (A-C) A/Hong Kong/1/1968 (H3), lane 1;A/Beijing/47/1992 (H3), lane 2; A/Pan/2007/1999 (H3), lane 3;A/Brisbane/10/2007 (H3), lane 4; A/New Caledonia20/1999 (H1), lane 5;A/DK/1964 (H4), lane 6; A/TKY/1963 (H7), lane 7.

FIG. 4. Monoclonal antibodies 7A7 and 12D1 inhibit low-pH fusion ofInfluenza virus strain A/Hong Kong/1/1968 (H3) hemagglutinin as measuredby red blood cell fusion assay. Monoclonal antibody 1A7 (IgG) isspecific for the Influenza A virus protein NS 1 and does not affectviral fusion.

FIG. 5. Mice injected with monoclonal antibody 12D1 one hour prior tochallenge with Influenza virus strain X31 survive longer than miceinjected with PBS alone. X31 is a chimeric virus expressing thehemagglutinin and neuraminidase proteins from A/Hong Kong/1/1968 (H3N2)on an A/PR/8 background (mouse-adapted H1N1 virus).

FIG. 6. Phylogenetic relationships among Influenza virus HA subtypesfrom Sui et al., 2009, Nat. Struct. Mol. Biol 16(3): 265-273.

FIG. 7. Passive transfer of monoclonal antibodies 12D1 and 39A4 resultsin decreased weight loss in mice challenged with Influenza A virusstrain A/Hong Kong/1/1968 (H3) as compared to mice administered PBS,rather than antibody.

FIG. 8. Diagram of fusion protein encoded by nucleic acid constructs:hemagglutinin truncation mutants fused to green fluorescent protein.

FIG. 9. Western blot assessing the ability of the monoclonal antibody12D1 to bind to fragments of the hemagglutinin protein of Influenza Avirus strain A/Hong Kong/1/1968 (H3).

FIG. 10. Monoclonal antibodies 7A7, 12D1 and 39A4 react with H3hemagglutinin by Western blot. (A) MAb 12D1 binds the A/Pan/2007/1999hemagglutinin within the HA2 subunit. Monoclonal antibodies 7A7 and 39A4do not react with hemagglutinin under reducing conditions. (B)Monoclonal antibodies 7A7, 12D1 and 39A4 react with the A/HK/1/1968hemagglutinin under non-reducing conditions. Monoclonal antibodies 7A7(lane 1) and 39A4 (lane 3) bind HA trimer complexes. mAb 12D1 (lane 2)binds HA trimer complexes and HA0.

FIG. 11. Reactivity of anti-H3 mAbs by ELISA. (A) Monoclonal antibodies7A7 and 39A4 react with purified A/HK/1968 (H3) virus. (B) Monoclonalantibodies 7A7, 12D1 and 39A4 react with purified A/Alabama/1981 (H3)virus. Monoclonal antibody XY102 is specific for the hemagglutinin ofA/HK/1968 virus.

FIG. 12. Anti-H3 monoclonal antibodies in microneutralization assay.Neutralization of virus expressing the HA from either (A) A/HongKong/1/1968 virus or (B) A/Panama/2007/1999 virus by monoclonalantibodies 7A7, 12D1 and 39A4. Monoclonal antibody XY102 is specific forA/HK/1968 virus. Purified mouse IgG was used for the negative control.

FIG. 13. Activity of anti-H3 mabs in plaque reduction assay on MDCKcells. Monoclonal antibodies 7A7 (A), 12D1 (B) and 39A4 (C) neutralizeall H3 viruses tested by plaque reduction assay but not representativeH1, H4 or H7 viruses. Purified mouse IgG was used for the negativecontrol. The plaque reduction assays were performed multiple times andwith each new antibody preparation.

FIG. 14. Anti-H3 monoclonal antibodies protect against H3 virus in vivo.Mice were given 30 mg/kg mAb 7A7, 12D1, 39A4 or isotype control byintraperitoneal injection 1 hour prior, 24 hours post (12D1 only) or 48hours post (12D1 only) challenge with X31. N=5 per group.

FIG. 15. Treatment with anti-H3 monoclonal antibodies diminishes lungdamage associated with viral pneumonia caused by X31 virus. (A,B)Untreated (C,D) mice treated with monoclonal antibody 39A4 (E,F) micetreated with mAb 12D1. 40× magnification.

FIG. 16. Anti-H3 monoclonal antibodies protect against replication of H3virus in lungs. Mice were given 30 mg/kg monoclonal antibody12D1, 39A4or isotype control by intraperitoneal injection 1 hour prior toinfection with A/Georgia/1981 virus. Data represent lung titers fromgroups of 5 mice, 2 days post infection.

FIG. 17. Red blood cell fusion assay. Anti-H3 monoclonal antibodiesinhibit low-pH induced fusion of HK/68 hemagglutinin with chicken redblood cells. All monoclonal antibodies are negative forhemagglutinin-inhibition activity. Monoclonal antibody 1A7 is specificfor Influenza virus NS1 protein.

FIG. 18. MAb 12D1 reacts by Western blot with hemagglutinin truncationmutants. 12D1 makes dominant contacts with the HA2 subunit in the regionof amino acids 30 to 106 (H3 numbering (see, e.g., Wilson et al., Nature1981; 289(5796):366-73)). Diminished 12D1 binding without diminished GFPexpression in the HA2 76-184 and HA2 91-184 truncations along with lossof binding with the HA2 106-184 truncation suggests that the bindingepitope lies in the region from amino acids HA2 76-106. These 30 aminoacids fall within the membrane distal half of the long alpha-helix ofHA2.

FIG. 19. The deduced nucleotide sequences of the VH and VL chains of theantibody 7A7. Framework regions are shown in bold. CDR regions areunderlined.

FIG. 20. The deduced amino acid sequences of the VH and VL chains of theantibody 7A7. Framework regions are shown in bold. CDR regions areunderlined.

FIG. 21. The deduced nucleotide sequences of the VH and VL chains of theantibody 12D1. Framework regions are shown in bold. CDR regions areunderlined.

FIG. 22. The deduced amino acid sequences of the VH and VL chains of theantibody 12D1. Framework regions are shown in bold. CDR regions areunderlined.

FIG. 23. Reactivity of monoclonal antibody 66A6 by ELISA. (A) Monoclonalantibody 66A6 reacts with purified A/HK/1968 (H3) virus. (B) Monoclonalantibody 66A6 reacts with purified A/Panama/2007/1999 (H3) virus. (C)Monoclonal antibody 66A6 reacts with purified A/Brisbane/10/2007 (H3)virus.

FIG. 24. Red blood cell fusion assay. Monoclonal antibody 66A6 isnegative for hemagglutinin-inhibition activity against purifiedA/HK/1968 (H3) virus. Monoclonal antibody XY102 is specific forA/HK/1968 virus.

FIG. 25. Monoclonal antibody 66A6 reacts with the hemagglutinin proteinof A/HK/1968 (H3) virus by Western blot, but does not react with non-H3viruses or recombinantly-expressed non-H3 hemagglutinins.

FIG. 26. Activity of monoclonal antibody 66A6 in plaque reduction assayon MDCK cells. Monoclonal antibody 66A6 neutralizes A/Panama/2007/1999(H3) and A/Alabama/1/1981 (H3) Influenza viruses by plaque reductionassay.

FIG. 27. Passive transfer of monoclonal antibody 66A6 results indecreased weight loss in mice challenged with Influenza X31 virus ascompared to mice administered PBS, rather than antibody. “M” representmouse.

FIG. 28. The deduced nucleotide sequences of the VH and VL chains of theantibody 66A6. Framework regions are shown in bold. CDR regions areunderlined.

FIG. 29. The deduced amino acid sequences of the VH and VL chains of theantibody 66A6. Framework regions are shown in bold. CDR regions areunderlined.

FIG. 30. Immunization strategy for cross-reactive anti-H1/H3 antibodies.

FIG. 31. Antibodies 1, 2, and 3 recognize the hemagglutinin protein ofthree H1N1 viruses, namelyA/USSR/77 (H1), A/Texas/91 (H1), andA/California/09 (H1), by immunofluorescence. Antibody 4 recognizes thehemagglutinin protein of two H3N2 viruses, namely A/HK/1968 (H3) virusand A/NY/08 (H3) virus, by immunofluorescence.

FIG. 32. Western blot results for Antibodies 1, 3, and 4: Antibodies 1and 3 do not recognize hemagglutinin protein under denaturing andreducing conditions. Antibody 4 recognizes the hemagglutinin protein ofH3 viruses under denaturing and reducing conditions.

FIG. 33. Immunization strategy for cross-reactive anti-H1 antibodies.

FIG. 34. Reactivity of potential clones from one hybridoma generatedusing the immunization strategy for cross-reactive anti-H1 antibodies byELISA.

5. DETAILED DESCRIPTION 5.1 Generation of Antibodies

Provided herein are methods for generating monoclonal antibodies thatbind to an Influenza virus antigen. Such monoclonal antibodies may bindto an Influenza virus antigen on an Influenza virus particle, e.g.,hemagglutinin. In a specific embodiment, the monoclonal antibodies bindto an Influenza virus particle. In another specific embodiment, themonoclonal antibodies selectively bind to hemagglutinin expressed byone, two, three or more strains of Influenza virus relative to anon-Influenza virus hemagglutinin antigen as assessed by techniquesknown in the art, e.g., ELISA, Western blot, FACs or BIACore. In otherwords, the monoclonal antibodies bind to hemagglutinin expressed by one,two, three or more strains of Influenza virus with a higher affinitythan a non-Influenza virus hemagglutinin antigen as assessed bytechniques known in the art, e.g., ELISA, Western blot, FACs or BIACore.

In a specific embodiment, a monoclonal antibody that binds to anInfluenza virus antigen, which is generated in accordance with a methodprovided herein, neutralizes a strain(s) of Influenza virus. In anotherspecific embodiment, a monoclonal antibody that binds to an Influenzavirus antigen, which is generated in accordance with a method providedherein, neutralizes antigenically distinct strains of Influenza virus(i.e., a cross-reactive neutralizing monoclonal antibody), as measuredby an assay known to those of skill in the art, e.g., a hemagglutinationinhibition assay. The antigenically distinct strains of Influenza virusmay be of the same subtype or a different subtype as determined by,e.g., the phylogenetic relationships among hemagglutinin and/orneuraminidase subtypes. For example, a monoclonal antibody generated inaccordance with a method provided herein may neutralize two, three ormore strains of the same Influenza virus hemagglutinin subtype (e.g.,the H3 subtype). In addition, a monoclonal antibody generated inaccordance with a method provided herein may neutralize strains ofdifferent Influenza virus hemagglutinin subtypes (e.g., Influenza virusstrains of the H1 and H3 subtypes) or may neutralize Influenza virushemagglutinin subtypes of 1, 2, or more clusters. A monoclonal antibodygenerated in accordance with a method provided herein may neutralize anInfluenza virus as a result of:

inhibiting or reducing the binding of the Influenza virus to a hostcell; and/or inhibiting or reducing the fusion of the Influenza viruswith a host cell.

In a specific embodiment, a monoclonal antibody generated in accordancewith a method described herein binds to amino acid residues within therange of 330 to 513, 359 to 513, 360 to 513, 375 to 513, 390 to 513,and/or 405 to 513 of a hemagglutinin polypeptide numbered according tothe HA0 polypeptide of the H3 subtype of Influenza virus. In anotherembodiment, a monoclonal antibody generated in accordance with a methoddescribed herein binds to amino acid residues within the range of 1-184,16-184, 30-184, 31-184, 46-184, 61-184, 70-110, 76-106, and/or 76-184 ofa hemagglutinin polypeptide numbered according to the classic H3 subtypenumbering system (see, Wilson I A, Skehel J J, Wiley DC (1981) Structureof the haemagglutinin membrane glycoprotein of influenza virus at 3 Aresolution. Nature 289:366-373 for classic H3 subtype numbering system).

In a specific embodiment an antibody generated in accordance with amethod described herein binds to the HA2 region of the hemagglutininpolypeptide of the Influenza virus strain A/Hong Kong/1/1968 (H3) (seeGenbank Accession No. AAK51718 for the amino acid sequence of thehemagglutinin polypeptide of A/Hong Kong/1/1968 (H3)). In anotherspecific embodiment, an antibody generated in accordance with a methoddescribed herein binds to the long alpha-helix of HA2 of an Influenzavirus (e.g., the hemagglutinin polypeptide of the Influenza virus strainA/Hong Kong/1/1968 (H3)). In a specific embodiment, an antibodygenerated in accordance with a method described herein binds to the longalpha-helix of the hemagglutinin polypeptide of the Influenza virusstrain A/Hong Kong/1/1968 (H3) (i.e., amino acids 76-130, numberedaccording to the classic H3 subtype numbering system), i.e., theantibody binds an epitope within the following amino acid sequence:RIQDLEKYVEDTKIDLWSYNAELLVALENQHTIDLTDSEMNKLFEKTRRQLRENA (SEQ ID NO:125).In another specific embodiment, a monoclonal antibody generated inaccordance with a method described herein binds to amino acid residueswithin the range of 304 to 513, 330 to 513, 345 to 513, 360 to 513, 375to 513, 390 to 513, and/or 405-513 of the hemagglutinin polypeptide ofthe Influenza virus strain A/Hong Kong/1/1968 (H3). In another specificembodiment, a monoclonal antibody generated in accordance with a methoddescribed herein binds to amino acid residues within the range of 330 to513, 359 to 513, 360 to 513, 375 to 513, 390 to 513, and/or 405 to 513of the hemagglutinin polypeptide of the Influenza virus strain A/HongKong/1/1968 (H3) numbered according to the HA0 polypeptide of the H3subtype of Influenza virus. In another specific embodiment, a monoclonalantibody generated in accordance with a method described herein binds toamino acid residues within the range of 1-184, 16-184, 30-184, 31-184,46-184, 61-184, 70-110, 76-106, and/or 76-184 of the hemagglutininpolypeptide of the Influenza virus strain A/Hong Kong/1/1968 (H3)numbered according to the classic H3 subtype numbering system (see,Wilson I A, Skehel J J, Wiley DC (1981) Structure of the haemagglutininmembrane glycoprotein of influenza virus at 3 A resolution. Nature289:366-373 for classic H3 subtype numbering system). In anotherspecific embodiment, a monoclonal antibody generated in accordance witha method described herein binds to an epitope in the hemagglutininpolypeptide of A/Hong Kong/1/1968 (H3) located within amino acids 405 to513 of the hemagglutinin polypeptide (i.e., within amino acids 76-183 inthe classic H3 subtype numbering system), i.e., the antibody binds anepitope within the following amino acid sequence: RIQDLEKYVE DTKIDLWSYNAELLVALENQ HTIDLTDSEM NKLFEKTRRQ LRENAEDMGN GCFKIYHKCD NACIESIRNGTYDHDVYRDE ALNNRFQIKG VELKSGYKD (SEQ ID NO:1).

In one aspect, a method for generating a monoclonal antibody that bindsto an Influenza virus antigen involves the administration of two, three,four or more immunogenic compositions to a non-human subject with theadministration of each immunogenic composition separated by a certainamount of time (e.g., about 2 to 4 weeks, about 2 to 6 weeks, about 4 to6 weeks, about 2 to 8 weeks or about 4 to 8 weeks), wherein eachimmunogenic composition comprises an inactivated Influenza virus, anattenuated Influenza virus (e.g., a live Influenza virus that has beenattenuated), a live Influenza virus other than an attenuated Influenzavirus (e.g., naturally occurring Influenza virus), an antigen derived orobtained from an Influenza virus, or a nucleic acid encoding an antigenderived or obtained from an Influenza virus, and wherein one immunogeniccomposition differs from another immunogenic composition in that theInfluenza virus, or the Influenza virus from which the antigen or thenucleic acid sequence encoding the antigen is derived or obtained areantigenically distinct. In one embodiment, such immunogenic compositionsdiffer from each other because at a minimum the Influenza virus, or theInfluenza virus from which the antigen or the nucleic acid sequenceencoding the antigen is derived or obtained that is included in eachcomposition are from antigenically distinct strains of one subtype(e.g., the H3 subtype). In another embodiment, such immunogeniccompositions differ from each other because at a minimum the Influenzavirus, or the Influenza virus from which the antigen or the nucleic acidsequence encoding the antigen is derived or obtained that is included ineach composition are from antigenically distinct strains of two, three,four, five or more subtypes (e.g., the H1 and H3 subtypes). A certainperiod of time after the administration of the last immunogeniccomposition (e.g., about 2 to 5 days, about 5 to 10 days, or about 8 to14 days), cells which can be used to produce hybridomas, e.g.,splenocytes or lymph node cells, may be harvested from the subject forthe production of hybridomas. Any technique known in the art may be usedto produce hybridomas (see, for example, the techniques taught in Harlowet al., Antibodies: A Laboratory Manual, (Cold Spring Harbor LaboratoryPress, 2nd ed. 1988); Hammerling, et al., in: Monoclonal Antibodies andT-Cell Hybridomas 563 681 (Elsevier, N.Y., 1981) (said referencesincorporated by reference in their entireties)). Supernatants fromhybridomas generated may be screened for binding to different strains ofInfluenza virus of the same and/or different subtypes as well asneutralization activity in a microneutralization assay such as describedin Example 6 infra. Monoclonal antibodies may then be isolated from thehybridomas. In a specific embodiment, a monoclonal antibody that bindsto and neutralizes two, three or more strains of Influenza virus of thesame subtype and/or different subtypes is generated in accordance withsuch a method. In some embodiments, the immunogenic compositioncomprises more than one inactivated Influenza virus, attenuatedInfluenza virus (e.g., a live Influenza virus that has been attenuated),live Influenza virus other than an attenuated Influenza virus, antigenderived or obtained from an Influenza virus, or nucleic acid encoding anantigen derived or obtained from an Influenza virus.

In one embodiment, a method for generating a monoclonal antibody thatbinds to an Influenza virus antigen comprises: (i) immunizing anon-human subject (e.g., a mouse, rabbit, rat, guinea pig, etc.) with aninactivated first Influenza virus, an attenuated first Influenza virus,a live first Influenza virus other than an attenuated Influenza virus,an antigen (e.g., hemagglutinin) derived or obtained from a firstInfluenza virus, or a nucleic acid encoding an antigen derived orobtained from a first Influenza virus; (ii) after a specified period oftime, immunizing the subject with an inactivated second Influenza virus,an attenuated second Influenza virus, a live second Influenza virusother than an attenuated Influenza virus, an antigen (e.g.,hemagglutinin) derived or obtained from a second Influenza virus, or anucleic acid encoding an antigen derived or obtained from a secondInfluenza virus, wherein the second Influenza virus is antigenicallydistinct from the first Influenza virus; and (iii) after a specifiedperiod of time, generating B-cell hybridomas from the subject andfurther selecting for hybridoma clones that express monoclonalantibodies that bind to the Influenza virus antigen. In certainembodiments, the method comprises selecting hybridoma clones thatexpress a monoclonal antibody that binds to the Influenza virus antigen.In specific embodiments, the monoclonal antibodies are isolated from thehybridomas. In certain embodiments, before monoclonal antibodies areisolated, the hybridomas may be screened for binding to differentstrains of Influenza virus of the same and/or different subtypes as wellas neutralization activity in a microneutralization assay such asdescribed in Example 6 infra. In some embodiments, only monoclonalantibodies that bind to and neutralize different strains of Influenzavirus of the same and/or different subtypes are isolated. In certainembodiments, immunization (i) above involves the administration of anantigen or nucleic acid construct, and immunization (ii) involves theadministration of inactivated or attenuated Influenza virus. In otherembodiments, immunization (i) above involves the administration ofinactivated or attenuated Influenza virus, and immunization (ii)involves the administration of an antigen or nucleic acid construct. Ina specific embodiment, a monoclonal antibody generated in accordancewith such a method binds to and neutralizes two, three or more strainsof Influenza virus of the same subtype and/or different subtypes.

In another embodiment, a method for generating a monoclonal antibodythat binds to an Influenza virus antigen comprises: (i) immunizing anon-human subject (e.g., a mouse, rabbit, rat, guinea pig, etc.) with aninactivated first Influenza virus, an attenuated first Influenza virus,a live first Influenza virus other than an attenuated Influenza virus,an antigen (e.g., hemagglutinin) derived or obtained from a firstInfluenza virus, or nucleic acid encoding an antigen derived or obtainedfrom a first Influenza virus; (ii) after a specified period of time,immunizing the subject with an inactivated second Influenza virus, anattenuated second Influenza virus, a live second Influenza virus otherthan an attenuated Influenza virus, an antigen derived or obtained froma second Influenza virus, or a nucleic acid encoding an antigen from asecond Influenza virus, wherein the second Influenza virus isantigenically distinct from the first Influenza virus; (iii) after aspecified period of time, immunizing the subject with an inactivatedthird Influenza virus, an attenuated third Influenza virus, a live thirdInfluenza virus other than an attenuated Influenza virus, an antigenderived or obtained from a third Influenza virus, or a nucleic acidencoding an antigen derived or obtained from a third Influenza virus,wherein the third Influenza virus is antigenically distinct from thesecond and first Influenza viruses; and (iv) after a specified period oftime, generating B-cell hybridomas from the subject that expressmonoclonal antibodies that bind to an Influenza virus antigen. Incertain embodiments, the method comprises selecting hybridoma clonesthat express a monoclonal antibody that binds to the Influenza virusantigen. In specific embodiments, the monoclonal antibodies are isolatedfrom the hybridomas. In certain embodiments, before monoclonalantibodies are isolated, the hybridomas may be screened for binding todifferent strains of Influenza virus of the same and/or differentsubtypes as well as neutralization activity in a microneutralizationassay such as described in Example 6 infra. In some embodiments, onlymonoclonal antibodies that bind to and neutralize different strains ofInfluenza virus of the same and/or different subtypes are isolated. Incertain embodiments, the immunizations (i) and (ii) above involve theadministration of an antigen or nucleic acid construct, and immunization(iii) involves the administration of inactivated or attenuated Influenzavirus. In other embodiments, the immunizations (i) and (ii) aboveinvolve the administration of inactivated or attenuated Influenza virus,and immunization (iii) involves the administration of an antigen ornucleic acid construct. In a specific embodiment, a monoclonal antibodygenerated in accordance with such a method binds to and neutralizes two,three or more strains of Influenza virus of the same subtype and/ordifferent subtypes.

In another embodiment, a method for generating a monoclonal antibodythat binds to an Influenza virus antigen comprises: (i) immunizing anon-human subject (e.g., a mouse, rabbit, rat, guinea pig, etc.) with aninactivated first Influenza virus, an attenuated first Influenza virusother than an attenuated Influenza virus, a live first Influenza virus,an antigen (e.g., hemagglutinin) derived or obtained from a firstInfluenza virus, or a nucleic acid encoding an antigen obtained orderived from a first Influenza virus; (ii) after a specified period oftime, immunizing the subject with an inactivated second Influenza virus,an attenuated second Influenza virus, a live second Influenza virusother than an attenuated Influenza virus, an antigen derived or obtainedfrom a second Influenza virus, or a nucleic acid encoding an antigenderived or obtained from a second Influenza virus, wherein the secondInfluenza virus is antigenically distinct from the first Influenzavirus; (iii) after a specified period of time, immunizing the subjectwith an inactivated third Influenza virus, an attenuated third Influenzavirus, a live third Influenza virus other than an attenuated Influenzavirus, an antigen derived or obtained from a third Influenza virus, or anucleic acid encoding an antigen derived or obtained from a thirdInfluenza virus, wherein the third Influenza virus is antigenicallydistinct from the second and first Influenza viruses; (iv) after aspecified period of time, immunizing the subject with an inactivatedfourth Influenza virus, an attenuated fourth Influenza virus, a livefourth Influenza virus other than an attenuated Influenza virus, anantigen derived or obtained from a fourth Influenza virus, or a nucleicacid encoding an antigen derived or obtained from a fourth Influenzavirus, wherein the fourth Influenza virus is antigenically distinct fromthe third, second, and first Influenza viruses; and (v) after aspecified period of time, generating B-cell hybridomas from the subjectthat express monoclonal antibodies that bind to an Influenza virusantigen. In certain embodiments, the method comprises selectinghybridoma clones that express a monoclonal antibody that binds to anInfluenza virus antigen. In specific embodiments, the monoclonalantibodies are isolated from the hybridomas. In certain embodiments,before monoclonal antibodies are isolated, the hybridomas may bescreened for binding to different strains of Influenza virus of the sameand/or different subtypes as well as neutralization activity in amicroneutralization assay such as described in Example 6 infra. In someembodiments, only monoclonal antibodies that bind to and neutralizedifferent strains of Influenza virus of the same and/or differentsubtypes are isolated. In certain embodiments, the immunizations (i),(ii) and (iii) above involve the administration of an antigen or nucleicacid construct, and immunization (iv) involves the administration ofinactivated or attenuated Influenza virus. In other embodiments, theimmunizations (i), (ii) and (iii) above involve the administration ofinactivated or attenuated Influenza virus, and immunization (iv)involves the administration of an antigen or nucleic acid construct. Ina specific embodiment, a monoclonal antibody generated in accordancewith such a method binds to and neutralizes two, three or more strainsof Influenza virus of the same subtype and/or different subtypes.

Once a monoclonal antibody has been produced in accordance with themethods described herein, it can be screened for its ability to bind toInfluenza viruses using methods known in the art and described herein.The monoclonal antibodies produced in accordance with the methodsdescribed herein can also be tested for their ability to neutralizeInfluenza virus using methods known in the art, e.g.,microneutralization assay, plaque reduction assay, and/or cell fusionassay, and described herein (see Section 5.7 and Example 6, infra).

According to methods provided herein, the specified period of timebetween immunizations of a non-human subject (e.g., a mouse, rabbit,rat, guinea pig, etc.) with inactivated Influenza virus, attenuatedInfluenza virus, live Influenza virus (e.g., naturally occurringInfluenza virus), an antigen (e.g., hemagglutinin) derived or obtainedfrom an Influenza virus, or a nucleic acid encoding an antigen derivedor obtained from an Influenza virus can be any time period sufficient toallow the subject to generate an antibody response to the Influenzavirus or the Influenza virus antigen. In some embodiments, the specifiedperiod of time between immunizations is 1 week, 10 days, 12 days, 2weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 10 weeks,12 weeks, 14 weeks, 16 weeks, or greater than 16 weeks. In otherembodiments, the specified period of time between immunizations rangesfrom about 2-4 weeks, about 2-6 weeks, about 2-8 weeks, about 3-4 weeks,about 3-5 weeks, about 3-7 weeks, about 4-6 weeks, about 4-8 weeks,about 4-12 weeks, and/or about 4-16 weeks. In certain embodiments, thespecified period of time between immunizations is not 10 days.

In some embodiments, the specified period of time between the finalimmunization of the non-human subject (e.g., a mouse, rabbit, rat,guinea pig, etc.) and the generation of B-cell hybridomas is 1 day, 2days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 10 days, 12 days,14 days, or more than 14 days. In other embodiments, the specifiedperiod of time between the final immunization of the non-human subjectand the generation of B-cell hybridomas is about 1-3 days, about 2-5days, about 3-7 days, about 4-8 days, about 5-10 days, or about 7-14days. In certain embodiments, the specified period of time between thefinal immunization of the non-human subject (e.g., a mouse, rabbit, rat,guinea pig, etc.) and the generation of B-cell hybridomas is not 3 days.

In a specific embodiment, a method for generating a monoclonal antibodythat binds to an Influenza virus antigen comprises: (i) immunizing amouse with a nucleic acid encoding hemagglutinin from Influenza A virusstrain A/Hong Kong/1/1968 (H3); (ii) after three weeks, immunizing themouse with a nucleic acid encoding hemagglutinin from Influenza A virusstrain A/Alabama/1/1981 (H3); (iii) after three weeks, immunizing themouse with a nucleic acid encoding hemagglutinin from Influenza A virusstrain A/Beijing/47/1992 (H3); (iv) after three weeks, immunizing themouse with Influenza A virus strain A/Wyoming/3/2003 (H3); and (v) afterthree days, generating hybridomas from splenocytes harvested from themouse. In specific embodiments, the monoclonal antibodies areharvested/isolated from hybridoma supernatants. In certain embodiments,before monoclonal antibodies are isolated, the hybridomas may bescreened for binding to different strains of Influenza virus of the sameand/or different subtypes as well as neutralization activity in amicroneutralization assay such as described in Example 6 infra. In someembodiments, only monoclonal antibodies that bind to and neutralizedifferent strains of Influenza virus of the same and/or differentsubtypes are isolated. In a specific embodiment, a monoclonal antibodygenerated in accordance with such a method binds to and neutralizes two,three or more strains of Influenza virus of the same subtype and/ordifferent subtypes.

In a specific embodiment, a method for generating a monoclonal antibodythat binds to an Influenza virus antigen comprises: (i) immunizing amouse with a nucleic acid encoding hemagglutinin from Influenza A virusstrain A/Hong Kong/1/1968 (H3); (ii) after three weeks, immunizing themouse with a nucleic acid encoding hemagglutinin from Influenza A virusstrain A/USSR/92/77 (H1); (iii) after three weeks, immunizing the mousewith a nucleic acid encoding hemagglutinin from Influenza A virus strainA/California/1/88 (H3); (iv) after three weeks, immunizing the mousewith Influenza A virus strain A/California/04/09 (H1); (v) after threeweeks, immunizing the mouse with a composition comprising Influenza Avirus strain A/Brisbane/59/07-like (H1) and Influenza A virus strainA/Brisbane/10/07-like (H3); and (vi) after three days, generatinghybridomas from splenocytes harvested from the mouse. In specificembodiments, the monoclonal antibodies are harvested/isolated fromhybridoma supernatants. In certain embodiments, before monoclonalantibodies are isolated, the hybridomas may be screened for binding todifferent strains of Influenza virus of the same and/or differentsubtypes as well as neutralization activity in a microneutralizationassay such as described in Example 6 infra. In some embodiments, onlymonoclonal antibodies that bind to and neutralize different strains ofInfluenza virus of the same and/or different subtypes are isolated. In aspecific embodiment, a monoclonal antibody generated in accordance withsuch a method binds to and neutralizes two, three or more strains ofInfluenza virus of the same subtype and/or different subtypes.

In a specific embodiment, a method for generating a monoclonal antibodythat binds to an Influenza virus antigen comprises: (i) immunizing amouse with a nucleic acid encoding hemagglutinin from Influenza A virusstrain A/South Carolina/1918 (H1); (ii) after three weeks, immunizingthe mouse with a nucleic acid encoding hemagglutinin from Influenza Avirus strain A/USSR/92/77 (H1); (iii) after three weeks, immunizing themouse with a nucleic acid encoding hemagglutinin from Influenza A virusstrain A/California/04/09 (H1); (iv) after three weeks, immunizing themouse with Influenza A virus strain A/Brisbane/59/07-like (H1); and (v)after three days, generating hybridomas from splenocytes harvested fromthe mouse. In specific embodiments, the monoclonal antibodies areharvested/isolated from hybridoma supernatants. In certain embodiments,before monoclonal antibodies are isolated, the hybridomas may bescreened for binding to different strains of Influenza virus of the sameand/or different subtypes as well as neutralization activity in amicroneutralization assay such as described in Example 6 infra. In someembodiments, only monoclonal antibodies that bind to and neutralizedifferent strains of Influenza virus of the same and/or differentsubtypes are isolated. In a specific embodiment, a monoclonal antibodygenerated in accordance with such a method binds to and neutralizes two,three or more strains of Influenza virus of the same subtype and/ordifferent subtypes.

In certain embodiments, the non-human subjects administered animmunogenic composition(s) in accordance with the methods describedherein are transgenic animals (e.g., transgenic mice) that are capableof producing human antibodies. Human antibodies can be produced usingtransgenic mice which are incapable of expressing functional endogenousimmunoglobulins, but which can express human immunoglobulin genes. Forexample, the human heavy and light chain immunoglobulin gene complexesmay be introduced randomly or by homologous recombination into mouseembryonic stem cells. Alternatively, the human variable region, constantregion, and diversity region may be introduced into mouse embryonic stemcells in addition to the human heavy and light chain genes. The mouseheavy and light chain immunoglobulin genes may be renderednon-functional separately or simultaneously with the introduction ofhuman immunoglobulin loci by homologous recombination. In particular,homozygous deletion of the JH region prevents endogenous antibodyproduction. The modified embryonic stem cells are expanded andmicroinjected into blastocysts to produce chimeric mice. The chimericmice are then bred to produce homozygous offspring which express humanantibodies. The human immunoglobulin transgenes harbored by thetransgenic mice rearrange during B cell differentiation, andsubsequently undergo class switching and somatic mutation. Thus, usingsuch a technique, it is possible to produce therapeutically useful IgG,IgA, IgM and IgE antibodies. For an overview of this technology forproducing human antibodies, see Lonberg and Huszar, Int. Rev. Immunol.13:65-93 (1995). For a detailed discussion of this technology forproducing human antibodies and human monoclonal antibodies and protocolsfor producing such antibodies, see, e.g., PCT publications WO 98/24893;WO 92/01047; WO 96/34096; WO 96/33735; European Patent No. 0 598 877;U.S. Pat. Nos. 5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016;5,545,806; 5,814,318; 5,885,793; 5,916,771; and 5,939,598, which areincorporated by reference herein in their entirety. Companies such asAbgenix, Inc. (Freemont, Calif.), Genpharm (San Jose, Calif.), andMedarex, Inc. (Princeton, N.J.) can be engaged to provide humanantibodies directed against a selected antigen.

In addition, the non-human subjects administered an immunogeniccomposition(s) described herein may be transplanted with humanperipheral blood leukocytes, splenocytes, or bone marrow (e.g., TriomaTechniques XTL) so that human antibodies that bind to an Influenza virusantigen are generated.

The steps of the methods provided herein for generating monoclonalantibodies are not limited to immunization with any particular number ofInfluenza virus strains and the immunization of the non-human subjectcan be repeated using any number of Influenza virus strains that areantigenically distinct from one another, e.g., 2 antigenically distinctInfluenza virus strains, 3 antigenically distinct Influenza virusstrains, 4 antigenically distinct Influenza virus strains, 5antigenically distinct Influenza virus strains, or 6 or moreantigenically distinct Influenza virus strains.

In certain embodiments, the antigenically distinct Influenza virusstrains used in accordance with the methods provided herein forgenerating monoclonal antibodies are selected based on the difference intime between the emergence of the Influenza virus strains. For example,Influenza viruses of the same subtype are likely to be antigenicallydistinct from one another as the difference in time between theiremergence becomes greater, i.e., an Influenza A virus of subtype H3 thatemerged in 1960 would have a high likelihood of being antigenicallydistinct from an Influenza A virus of subtype H3 that emerged in 1980.In a specific embodiment, antigenically distinct Influenza virus strainsinclude strains that have emerged over a period of about 10 years, about15 years, about 20 years, about 25 years, about 30 years, about 40 yearsor about 50 years. In another specific embodiment, antigenicallydistinct Influenza virus strains include strains that have emerged overa period of about 10 to 100 years, about 10 to 75 years, about 10 to 50years, about 10 to 40 years, about 10 to 30 years, about 10 to 25 years,or about 10 to 20 years. In certain embodiments, Influenza virusstrains, or Influenza virus antigens or nucleic acids encoding Influenzavirus antigens used in accordance with the methods provided herein forgenerating monoclonal antibodies are selected from virus strains thathave emerged about every 5 years, about every 10 years, or about every20 years over a 40 year period, or over a 40 to 50 year period. In otherembodiments, Influenza virus strains, or Influenza virus antigens ornucleic acids encoding Influenza virus antigen used in accordance withthe methods provided herein for generating monoclonal antibodies areselected from virus strains that have emerged about every 5 years, aboutevery 10 years, about every 20 years, about every 25 years, or aboutevery 30 years over a 75 to 100 year period.

In certain embodiments, Influenza virus strains, or Influenza virusantigens or nucleic acids encoding Influenza virus antigen used inaccordance with the methods provided herein for generating monoclonalantibodies are selected from virus strains that have emerged over aperiod of about 7 years. In other embodiments, Influenza virus strains,or Influenza virus antigens or nucleic acids encoding Influenza virusantigen used in accordance with the methods provided herein forgenerating monoclonal antibodies are not selected from virus strainsthat have emerged over a period of about 7 years. In certainembodiments, Influenza virus strains, or Influenza virus antigens ornucleic acids encoding Influenza virus antigen used in accordance withthe methods provided herein for generating monoclonal antibodies areselected from virus strains that have emerged over a period of about 6-8years. In other embodiments, Influenza virus strains, or Influenza virusantigens or nucleic acids encoding Influenza virus antigen used inaccordance with the methods provided herein for generating monoclonalantibodies are not selected from virus strains that have emerged over aperiod of about 6-8 years.

In other embodiments, the antigenically distinct Influenza virus strainsused in accordance with the methods provided herein for generatingmonoclonal antibodies are selected from viruses that emerged at oraround the same time, e.g., within the same year, but are antigenicallydistinct from each other.

In certain embodiments, the Influenza viruses, or the Influenza virusesthat antigens or nucleic acids encoding antigens are derived or obtainedfrom are strains of Influenza A viruses. In one embodiment, theInfluenza viruses, or the Influenza viruses that antigens or nucleicacids encoding antigens are derived or obtained from are strains ofInfluenza A viruses from a single subtype. In another embodiment, theInfluenza viruses, or the Influenza viruses that antigens or nucleicacids encoding antigens are derived or obtained from are strains ofInfluenza A viruses from two, three or more subtypes. In anotherembodiment, the Influenza viruses, or the Influenza viruses thatantigens or nucleic acids encoding antigens are derived or obtained fromare strains of Influenza A viruses from one, two, or more clusters(e.g., the H1 cluster of H1a Influenza viruses (H2, H5, H1, and H6) andH1b Influenza viruses (H13, H16, and H11), the H9 cluster of Influenzaviruses (H8, H12, and H9), the H3 cluster of Influenza viruses (H4, H14,and H3), or the H7 cluster of Influenza viruses (H15, H7, and H10)).Non-limiting examples of Influenza A viruses include subtype H10N4,subtype H10N5, subtype H10N7, subtype H10N8, subtype H10N9, subtypeH11N1, subtype H11N13, subtype H11N2, subtype H11N4, subtype H11N6,subtype H11N8, subtype H11N9, subtype H12N1, subtype H12N4, subtypeH12N5, subtype H12N8, subtype H13N2, subtype H13N3, subtype H13N6,subtype H13N7, subtype H14N5, subtype H14N6, subtype H15N8, subtypeH15N9, subtype H16N3, subtype H1N1, subtype H1N2, subtype H1N3, subtypeH1N6, subtype H1N9, subtype H2N1, subtype H2N2, subtype H2N3, subtypeH2N5, subtype H2N7, subtype H2N8, subtype H2N9, subtype H3N1, subtypeH3N2, subtype H3N3, subtype H3N4, subtype H3N5, subtype H3N6, subtypeH3N8, subtype H3N9, subtype H4N1, subtype H4N2, subtype H4N3, subtypeH4N4, subtype H4N5, subtype H4N6, subtype H4N8, subtype H4N9, subtypeH5N1, subtype H5N2, subtype H5N3, subtype H5N4, subtype H5N6, subtypeH5N7, subtype H5N8, subtype H5N9, subtype H6N1, subtype H6N2, subtypeH6N3, subtype H6N4, subtype H6N5, subtype H6N6, subtype H6N7, subtypeH6N8, subtype H6N9, subtype H7N1, subtype H7N2, subtype H7N3, subtypeH7N4, subtype H7N5, subtype H7N7, subtype H7N8, subtype H7N9, subtypeH8N4, subtype H8N5, subtype H9N1, subtype H9N2, subtype H9N3, subtypeH9N5, subtype H9N6, subtype H9N7, subtype H9N8, and subtype H9N9.

Specific examples of strains of Influenza A virus include, but are notlimited to: A/sw/Iowa/15/30 (H1N1); A/WSN/33 (H1N1); A/eq/Prague/1/56(H7N7); A/PR/8/34; A/mallard/Potsdam/178-4/83 (H2N2); A/herringgull/DE/712/88 (H16N3); A/sw/Hong Kong/168/1993 (H1N1);A/mallard/Alberta/211/98 (H1N1); A/shorebird/Delaware/168/06 (H16N3);A/sw/Netherlands/25/80 (H1N1); A/sw/Germany/2/81 (H1N1);A/sw/Hannover/1/81 (H1N1); A/sw/Potsdam/1/81 (H1N1); A/sw/Potsdam/15/81(H1N1); A/sw/Potsdam/268/81 (H1N1); A/sw/Finistere/2899/82 (H1N1);A/sw/Potsdam/35/82 (H3N2); A/sw/Cote d′Armor/3633/84 (H3N2);A/sw/Gent/1/84 (H3N2); A/sw/Netherlands/12/85 (H1N1);A/sw/Karrenzien/2/87 (H3N2); A/sw/Schwerin/103/89 (H1N1);A/turkey/Germany/3/91 (H1N1); A/sw/Germany/8533/91 (H1N1);A/sw/Belgium/220/92 (H3N2); A/sw/GentN230/92 (H1N1); A/sw/Leipzig/145/92(H3N2); A/sw/Re220/92 hp (H3N2); A/sw/Bakum/909/93 (H3N2);A/sw/Schleswig-Holstein/1/93 (H1N1); A/sw/Scotland/419440/94 (H1N2);A/sw/Bakum/5/95 (H1N1); A/sw/Best/5C/96 (H1N1); A/sw/England/17394/96(H1N2); A/sw/Jena/5/96 (H3N2); A/sw/Oedenrode/7C/96 (H3N2);A/sw/Lohne/1/97 (H3N2); A/sw/Cote d′Armor/790/97 (H1N2);A/sw/Bakum/1362/98 (H3N2); A/sw/Italy/1521/98 (H1N2);A/sw/Italy/1553-2/98 (H3N2); A/sw/Italy/1566/98 (H1N1);A/sw/Italy/1589/98 (H1N1); A/sw/Bakum/8602/99 (H3N2); A/sw/Cotesd′Armor/604/99 (H1N2); A/sw/Cote d′Armor/1482/99 (H1N1);A/sw/Gent/7625/99 (H1N2); A/Hong Kong/1774/99 (H3N2); A/sw/HongKong/5190/99 (H3N2); A/sw/Hong Kong/5200/99 (H3N2); A/sw/HongKong/5212/99 (H3N2); A/sw/Ille et Villaine/1455/99 (H1N1);A/sw/Italy/1654-1/99 (H1N2); A/sw/Italy/2034/99 (H1N1);A/sw/Italy/2064/99 (H1N2); A/sw/Berlin/1578/00 (H3N2);A/sw/Bakum/1832/00 (H1N2); A/sw/Bakum/1833/00 (H1N2); A/sw/Coted′Armor/800/00 (H1N2); A/sw/Hong Kong/7982/00 (H3N2); A/sw/Italy/1081/00(H1N2); A/sw/Belzig/2/01 (H1N1); A/sw/Belzig/54/01 (H3N2); A/sw/HongKong/9296/01 (H3N2); A/sw/Hong Kong/9745/01 (H3N2); A/sw/Spain/33601/01(H3N2); A/sw/Hong Kong/1144/02 (H3N2); A/sw/Hong Kong/1197/02 (H3N2);A/sw/Spain/39139/02 (H3N2); A/sw/Spain/42386/02 (H3N2);A/Switzerland/8808/2002 (H1N1); A/sw/Bakum/1769/03 (H3N2);A/sw/Bissendorf/IDT1864/03 (H3N2); A/sw/Ehren/IDT2570/03 (H1N2);A/sw/Gescher/IDT2702/03 (H1N2); A/sw/Haselünne/2617/03 hp (H1N1);A/sw/Löningen/IDT2530/03 (H1N2); A/sw/IVD/IDT2674/03 (H1N2);A/sw/Nordkirchen/IDT1993/03 (H3N2); A/sw/Nordwalde/IDT2197/03 (H1N2);A/sw/Norden/IDT2308/03 (H1N2); A/sw/Spain/50047/03 (H1N1);A/sw/Spain/51915/03 (H1N1); A/sw/Vechta/2623/03 (H1N1);A/swNisbek/IDT2869/03 (H1N2); A/sw/Waltersdorf/IDT2527/03 (H1N2);A/sw/Damme/IDT2890/04 (H3N2); A/sw/Geldern/IDT2888/04 (H1N1);A/sw/Granstedt/IDT3475/04 (H1N2); A/sw/Greven/IDT2889/04 (H1N1);A/sw/Gudensberg/IDT2930/04 (H1N2); A/sw/Gudensberg/IDT2931/04 (H1N2);A/sw/Lohne/IDT3357/04 (H3N2); A/sw/Nortrup/IDT3685/04 (H1N2);A/sw/Seesen/IDT3055/04 (H3N2); A/sw/Spain/53207/04 (H1N1);A/sw/Spain/54008/04 (H3N2); A/sw/Stolzenau/IDT3296/04 (H1N2);A/sw/Wedel/IDT2965/04 (H1N1); A/sw/Bad Griesbach/IDT4191/05 (H3N2);A/sw/Cloppenburg/IDT4777/05 (H1N2); A/sw/Dotlingen/IDT3780/05 (H1N2);A/sw/Dotlingen/IDT4735/05 (H1N2); A/sw/Egglham/IDT5250/05 (H3N2);A/sw/Harkenblek/IDT4097/05 (H3N2); A/sw/Hertzen/IDT4317/05 (H3N2);A/sw/Krogel/IDT4192/05 (H1N1); A/sw/Laer/IDT3893/05 (H1N1);A/sw/Laer/IDT4126/05 (H3N2); A/sw/Merzen/IDT4114/05 (H3N2);A/sw/Muesleringen-S./IDT4263/05 (H3N2); A/sw/Osterhofen/IDT4004/05(H3N2); A/sw/Sprenge/IDT3805/05 (H1N2); A/sw/Stadtlohn/IDT3853/05(H1N2); A/swNoglarn/IDT4096/05 (H1N1); A/sw/Wohlerst/IDT4093/05 (H1N1);A/sw/Bad Griesbach/IDT5604/06 (H1N1); A/sw/Herzlake/IDT5335/06 (H3N2);A/sw/Herzlake/IDT5336/06 (H3N2); A/sw/Herzlake/IDT5337/06 (H3N2); andA/wild boar/Germany/R169/2006 (H3N2).

Other specific examples of strains of Influenza A virus include, but arenot limited to: A/Toronto/3141/2009 (H1N1); A/Regensburg/D6/2009 (H1N1);A/Bayern/62/2009 (H1N1); A/Bayern/62/2009 (H1N1); A/Bradenburg/19/2009(H1N1); A/Bradenburg/20/2009 (H1N1); A/Distrito Federal/2611/2009(H1N1); A/Mato Grosso/2329/2009 (H1N1); A/Sao Paulo/1454/2009 (H1N1);A/Sao Paulo/2233/2009 (H1N1); A/Stockholm/37/2009 (H1N1);A/Stockholm/41/2009 (H1N1); A/Stockholm/45/2009 (H1N1);A/swine/Alberta/OTH-33-1/2009 (H1N1); A/swine/Alberta/OTH-33-14/2009(H1N1); A/swine/Alberta/OTH-33-2/2009 (H1N1);A/swine/Alberta/OTH-33-21/2009 (H1N1); A/swine/Alberta/OTH-33-22/2009(H1N1); A/swine/Alberta/OTH-33-23/2009 (H1N1);A/swine/Alberta/OTH-33-24/2009 (H1N1); A/swine/Alberta/OTH-33-25/2009(H1N1); A/swine/Alberta/OTH-33-3/2009 (H1N1);A/swine/Alberta/OTH-33-7/2009 (H1N1); A/Beijing/502/2009 (H1N1);A/Firenze/10/2009 (H1N1); A/Hong Kong/2369/2009 (H1N1); A/Italy/85/2009(H1N1); A/Santo Domingo/572N/2009 (H1N1); A/Catalonia/385/2009 (H1N1);A/Catalonia/386/2009 (H1N1); A/Catalonia/387/2009 (H1N1);A/Catalonia/390/2009 (H1N1); A/Catalonia/394/2009 (H1N1);A/Catalonia/397/2009 (H1N1); A/Catalonia/398/2009 (H1N1);A/Catalonia/399/2009 (H1N1); A/Sao Paulo/2303/2009 (H1N1);A/Akita/1/2009 (H1N1); A/Castro/JXP/2009 (H1N1); A/Fukushima/1/2009(H1N1); A/Israel/276/2009 (H1N1); A/Israel/277/2009 (H1N1);A/Israel/70/2009 (H1N1); A/Iwate/1/2009 (H1N1); A/Iwate/2/2009 (H1N1);A/Kagoshima/1/2009 (H1N1); A/Osaka/180/2009 (H1N1); A/PuertoMontt/Bio87/2009 (H1N1); A/Sao Paulo/2303/2009 (H1N1); A/Sapporo/1/2009(H1N1); A/Stockholm/30/2009 (H1N1); A/Stockholm/31/2009 (H1N1);A/Stockholm/32/2009 (H1N1); A/Stockholm/33/2009 (H1N1);A/Stockholm/34/2009 (H1N1); A/Stockholm/35/2009 (H1N1);A/Stockholm/36/2009 (H1N1); A/Stockholm/38/2009 (H1N1);A/Stockholm/39/2009 (H1N1); A/Stockholm/40/2009 (H1N1;)A/Stockholm/42/2009 (H1N1); A/Stockholm/43/2009 (H1N1);A/Stockholm/44/2009 (H1N1); A/Utsunomiya/2/2009 (H1N1);A/WRAIR/0573N/2009 (H1N1); and A/Zhejiang/DTID-ZJU01/2009 (H1N1).

In certain embodiments, the Influenza viruses, or the Influenza virusesthat antigens or nucleic acids encoding antigens are derived or obtainedfrom are not subtype H3N2. In some embodiments, the Influenza viruses,or the Influenza viruses that antigens or nucleic acids encodingantigens are derived or obtained from are H3N2 strain A/Aichi/2/68, H3N2strain A/Victoria/3/75, or H3N2 strain A/Philippines/2/82. In otherembodiments, the Influenza viruses, or the Influenza viruses thatantigens or nucleic acids encoding antigens are derived or obtained fromare not H3N2 strain A/Aichi/2/68, H3N2 strain A/Victoria/3/75, or H3N2strain A/Philippines/2/82.

In other embodiments, the Influenza viruses, or the Influenza virusesthat antigens or nucleic acids encoding antigens are derived or obtainedfrom are A/Hong Kong/1/1968 (HK/68) (H3), A/Alabama/1/1981 (AL/81) (H3),A/Georgia/1981 (H3), A/Beijing/47/1992 (BJ/92) (H3), A/Wyoming/3/2003(H3), A/Wisconsin/67/2005 (WI/05) (H3), A/Brisbane/10/2007 (BR/07) (H3),A/New York/2008 (NY08) (H3), A/Texas/36/1991 (TX/91) (H1), A/NewCaledonia/20/99 (N.Cal/99) (H1), A/Duck/England/1962 (Dk/62) (H4),A/Turkey/England/1963 (Tky/63) (H7), A/Equine/Kentucky/2002 (e/KY/02)(H3), A/Ann Arbor/6/1960 (AA/60) (H2), A/Fort Monmouth/1/1947 (FM/47)(H1), A/USSR/92/77 (H1), A/California/1/88 (H3), A/California/04/09(H1), A/Brisbane/59/07-like (H1), A/Brisbane/10/07-like (H3) and/orA/South Carolina/1918 (H1).

There are currently 16 hemagglutinin subtypes of Influenza viruses thatfall into two different groups and any one, two or more of such subtypesmay be used in accordance with the methods provided herein. See FIG. 6for a table of the phylogenetic relationships among hemagglutininsubtypes. In a specific embodiment, the Influenza viruses, or theInfluenza viruses that antigens or nucleic acids encoding antigens arederived or obtained from strains of Influenza A viruses from a singlehemagglutinin subtype (e.g., H1 or H3). In a specific embodiment, theInfluenza viruses, or the Influenza viruses that antigens or nucleicacids encoding antigens are derived or obtained from are strains ofInfluenza A viruses from two, three or more hemagglutinin subtypes(e.g., H1 and H3). In another specific embodiment, the Influenzaviruses, or the Influenza viruses that antigens or nucleic acidsencoding antigens are derived or obtained from are strains of InfluenzaA viruses from one, two, or more clusters (e.g., the H1 cluster of H1aInfluenza viruses (H2, H5, H1, and H6) and H1b Influenza viruses (H13,H16, and H11), the H9 cluster of Influenza viruses (H8, H12, and H9),the H3 cluster of Influenza viruses (H4, H14, and H3), or the H7 clusterof Influenza viruses (H15, H7, and H10)).

In certain embodiments, the Influenza viruses, or the Influenza virusesthat antigens or nucleic acids encoding antigens are derived or obtainedare from strains of Influenza B viruses. Specific examples of InfluenzaB viruses include strain Aichi/5/88, strain Akita/27/2001, strainAkita/5/2001, strain Alaska/16/2000, strain Alaska/1777/2005, strainArgentina/69/2001, strain Arizona/146/2005, strain Arizona/148/2005,strain Bangkok/163/90, strain Bangkok/34/99, strain Bangkok/460/03,strain Bangkok/54/99, strain Barcelona/215/03, strain Beijing/15/84,strain Beijing/184/93, strain Beijing/243/97, strain Beijing/43/75,strain Beijing/5/76, strain Beijing/76/98, strain Belgium/WV106/2002,strain Belgium/WV107/2002, strain Belgium/WV109/2002, strainBelgium/WV114/2002, strain Belgium/WV122/2002, strain Bonn/43, strainBrazil/952/2001, strain Bucharest/795/03, strain Buenos Aires/161/00),strain Buenos Aires/9/95, strain Buenos Aires/SW16/97, strain BuenosAiresNL518/99, strain Canada/464/2001, strain Canada/464/2002, strainChaco/366/00, strain Chaco/R113/00, strain Cheju/303/03, strainChiba/447/98, strain Chongqing/3/2000, strain clinical isolate SA1Thailand/2002, strain clinical isolate SA10 Thailand/2002, strainclinical isolate SA100 Philippines/2002, strain clinical isolate SA101Philippines/2002, strain clinical isolate SA110 Philippines/2002),strain clinical isolate SA112 Philippines/2002, strain clinical isolateSA113 Philippines/2002, strain clinical isolate SA114 Philippines/2002,strain clinical isolate SA2 Thailand/2002, strain clinical isolate SA20Thailand/2002, strain clinical isolate SA38 Philippines/2002, strainclinical isolate SA39 Thailand/2002, strain clinical isolate SA99Philippines/2002, strain CNIC/27/2001, strain Colorado/2597/2004, strainCordoba/VA418/99, strain Czechoslovakia/16/89, strainCzechoslovakia/69/90, strain Daeku/10/97, strain Daeku/45/97, strainDaeku/47/97, strain Daeku/9/97, strain B/Du/4/78, strain B/Durban/39/98,strain Durban/43/98, strain Durban/44/98, strain B/Durban/52/98, strainDurban/55/98, strain Durban/56/98, strain England/1716/2005, strainEngland/2054/2005), strain England/23/04, strain Finland/154/2002,strain Finland/159/2002, strain Finland/160/2002, strainFinland/161/2002, strain Finland/162/03, strain Finland/162/2002, strainFinland/162/91, strain Finland/164/2003, strain Finland/172/91, strainFinland/173/2003, strain Finland/176/2003, strain Finland/184/91, strainFinland/188/2003, strain Finland/190/2003, strain Finland/220/2003,strain Finland/WV5/2002, strain Fujian/36/82, strain Geneva/5079/03,strain Genoa/11/02, strain Genoa/2/02, strain Genoa/21/02, strainGenova/54/02, strain Genova/55/02, strain Guangdong/05/94, strainGuangdong/08/93, strain Guangdong/5/94, strain Guangdong/55/89, strainGuangdong/8/93, strain Guangzhou/7/97, strain Guangzhou/86/92, strainGuangzhou/87/92, strain Gyeonggi/592/2005, strain Hannover/2/90, strainHarbin/07/94, strain Hawaii/10/2001, strain Hawaii/1990/2004, strainHawaii/38/2001, strain Hawaii/9/2001, strain Hebei/19/94, strainHebei/3/94), strain Henan/22/97, strain Hiroshima/23/2001, strain HongKong/110/99, strain Hong Kong/1115/2002, strain Hong Kong/112/2001,strain Hong Kong/123/2001, strain Hong Kong/1351/2002, strain HongKong/1434/2002, strain Hong Kong/147/99, strain Hong Kong/156/99, strainHong Kong/157/99, strain Hong Kong/22/2001, strain Hong Kong/22/89,strain Hong Kong/336/2001, strain Hong Kong/666/2001, strain HongKong/9/89, strain Houston/1/91, strain Houston/1/96, strainHouston/2/96, strain Hunan/4/72, strain Ibaraki/2/85, strainncheon/297/2005, strain India/3/89, strain India/77276/2001, strainIsrael/95/03, strain Israel/WV187/2002, strain Japan/1224/2005, strainJiangsu/10/03, strain Johannesburg/1/99, strain Johannesburg/96/01,strain Kadoma/1076/99, strain Kadoma/122/99, strain Kagoshima/15/94,strain Kansas/22992/99, strain Khazkov/224/91, strain Kobe/1/2002,strain, strain Kouchi/193/99, strain Lazio/1/02, strain Lee/40, strainLeningrad/129/91, strain Lissabon/2/90), strain Los Angeles/1/02, strainLusaka/270/99, strain Lyon/1271/96, strain Malaysia/83077/2001, strainMaputo/1/99, strain Mar del Plata/595/99, strain Maryland/1/01, strainMemphis/1/01, strain Memphis/12/97-MA, strain Michigan/22572/99, strainMie/1/93, strain Milano/1/01, strain Minsk/318/90, strain Moscow/3/03,strain Nagoya/20/99, strain Nanchang/1/00, strain Nashville/107/93,strain Nashville/45/91, strain Nebraska/2/01, strain Netherland/801/90,strain Netherlands/429/98, strain New York/1/2002, strain NIB/48/90,strain Ningxia/45/83, strain Norway/1/84, strain Oman/16299/2001, strainOsaka/1059/97, strain Osaka/983/97-V2, strain Oslo/1329/2002, strainOslo/1846/2002, strain Panama/45/90, strain Paris/329/90, strainParma/23/02, strain Perth/211/2001, strain Peru/1364/2004, strainPhilippines/5072/2001, strain Pusan/270/99, strain Quebec/173/98, strainQuebec/465/98, strain Quebec/7/01, strain Roma/1/03, strainSaga/S172/99, strain Seoul/13/95, strain Seoul/37/91, strainShangdong/7/97, strain Shanghai/361/2002), strain Shiga/T30/98, strainSichuan/379/99, strain Singapore/222/79, strain Spain/WV27/2002, strainStockholm/10/90, strain Switzerland/5441/90, strain Taiwan/0409/00,strain Taiwan/0722/02, strain Taiwan/97271/2001, strain Tehran/80/02,strain Tokyo/6/98, strain Trieste/28/02, strain Ulan Ude/4/02, strainUnited Kingdom/34304/99, strain USSR/100/83, strain Victoria/103/89,strain Vienna/1/99, strain Wuhan/356/2000, strain WV194/2002, strainXuanwu/23/82, strain Yamagata/1311/2003, strain Yamagata/K500/2001,strain Alaska/12/96, strain GA/86, strain NAGASAKI/1/87, strainTokyo/942/96, and strain Rochester/02/2001.

In certain embodiments, the Influenza viruses, or the Influenza virusesthat antigens or nucleic acids encoding antigens are derived or obtainedfrom are strains of Influenza C viruses. Specific examples of InfluenzaC viruses include strain Aichi/1/81, strain Ann Arbor/1/50, strainAomori/74, strain California/78, strain England/83, strain Greece/79,strain Hiroshima/246/2000, strain Hiroshima/252/2000, strain Hyogo/1/83,strain Johannesburg/66, strain Kanagawa/1/76, strain Kyoto/1/79, strainMississippi/80, strain Miyagi/1/97, strain Miyagi/5/2000, strainMiyagi/9/96, strain Nara/2/85, strain NewJersey/76, strainpig/Beijing/115/81, strain Saitama/3/2000), strain Shizuoka/79, strainYamagata/2/98, strain Yamagata/6/2000, strain Yamagata/9/96, strainBERLIN/1/85, strain ENGLAND/892/8, strain GREAT LAKES/1167/54, strainJJ/50, strain PIG/BEIJING/10/81, strain PIG/BEIJING/439/82), strainTAYLOR/1233/47, and strain C/YAMAGATA/10/81.

In certain embodiments, the Influenza viruses, or the Influenza virusesthat antigens or nucleic acids encoding antigens are derived or obtainedfrom are strains of Influenza A virus and strains of Influenza B virus.In some embodiments, the Influenza viruses, or the Influenza virusesthat antigens or nucleic acids encoding antigens are derived or obtainedfrom are strains of Influenza A virus, strains of Influenza B virus, andstrains of Influenza C virus.

In certain embodiments, the Influenza viruses, or the Influenza virusesthat antigens or nucleic acids encoding antigens are derived or obtainedfrom are treated with bromelain. In other embodiments, the Influenzaviruses, or the Influenza viruses that antigens or nucleic acidsencoding antigens are derived or obtained from are not treated withbromelain.

In a specific embodiment, the Influenza viruses administered to anon-human subject are isolated or purified. The Influenza virusesdescribed herein may be isolated and purified by any method known tothose of skill in the art. In one embodiment, the virus is removed fromcell culture and separated from cellular components, typically by wellknown clarification procedures, e.g., such as gradient centrifugationand column chromatography, and may be further purified as desired usingprocedures well known to those skilled in the art, e.g., plaque assays.

In a specific embodiment, antigens derived or obtained from a strain(s)of Influenza virus that are administered to a non-human subject areisolated. In another specific embodiment, nucleic acids encodingantigens derived or obtained from a strain(s) of Influenza virus thatare administered to a non-human subject are isolated.

An “isolated” nucleic acid, such as a cDNA molecule, can besubstantially free of other cellular material, or culture medium whenproduced by recombinant techniques, or substantially free of chemicalprecursors or other chemicals when chemically synthesized. The term“substantially free of cellular material” includes preparations ofnucleic acid in which the nucleic acid is separated from cellularcomponents of the cells from which it is isolated or recombinantlyproduced. Thus, nucleic acid that is substantially free of cellularmaterial includes preparations of nucleic acid having less than about30%, 20%, 10%, or 5% (by dry weight) of other nucleic acids. The term“substantially free of culture medium” includes preparations of nucleicacid in which the culture medium represents less than about 50%, 20%,10%, or 5% of the volume of the preparation. The term “substantiallyfree of chemical precursors or other chemicals” includes preparations inwhich the nucleic acid is separated from chemical precursors or otherchemicals which are involved in the synthesis of the nucleic acid. Inspecific embodiments, such preparations of the nucleic acid have lessthan about 50%, 30%, 20%, 10%, 5% (by dry weight) of chemical precursorsor compounds other than the nucleic acid of interest.

In accordance with the methods described herein, Influenza virus (live,inactivated or attenuated (e.g., a live Influenza virus that has beenattenuated)), antigens (e.g., hemagglutinin) derived or obtained fromInfluenza virus, or a nucleic acid encoding an antigen derived orobtained from an Influenza virus may be delivered to a non-human subjectby a variety of routes. Such routes include, but are not limited to,intranasal, intratracheal, oral, intradermal, transdermal,intramuscular, intraperitoneal, transdermal, intravenous, conjunctivaland subcutaneous routes. In a specific embodiment, the Influenza virus,antigen derived or obtained from Influenza virus, or a nucleic acidencoding an antigen derived or obtained from an Influenza virus isformulated in a composition containing excipients or carriers and thecomposition is administered to the non-human subject. Such compositionsare preferably suited for the route of administration to a non-humansubject.

In cases where Influenza virus, or a viral vector or viral-like particleis used to administer a nucleic acid encoding an antigen derived orobtained from an Influenza virus, it may be preferable to introduce thevirus, viral vector or viral-like particle via the natural route ofinfection for the virus, viral vector or viral-like particle.Alternatively, it may be preferable to introduce the viral vector orvirus-like particle via the natural route of infection of the Influenzavirus from which nucleic acid encoding the antigen is derived. Theability of an antigen, particularly a viral vector, to induce a vigoroussecretory and cellular immune response can be used advantageously. Forexample, infection of the respiratory tract by Influenza virus or aviral vector may induce a strong secretory immune response, for examplein the urogenital system, with concomitant protection against anInfluenza virus. In addition, in a preferred embodiment it may bedesirable to introduce the Influenza virus into the lungs by anysuitable route. Pulmonary administration can also be employed, e.g., byuse of an inhaler or nebulizer, and formulation with an aerosolizingagent for use as a spray.

The amount of Influenza virus (live, inactivated or attenuated (e.g., alive Influenza virus that has been attenuated)), antigen (e.g.,hemagglutinin) derived or obtained from an Influenza virus, or a nucleicacid encoding an antigen derived or obtained from an Influenza virusused to immunize a non-human subject in accordance with the methodsprovided herein for generating monoclonal antibodies can be determinedby methods known in the art. In certain embodiments, where the non-humansubject is administered Influenza virus (live, inactivated or attenuated(e.g., a live Influenza virus that has been attenuated)) or a viralvector containing a nucleic acid encoding an antigen derived or obtainedfrom an Influenza virus, approximately 10⁴ pfu, approximately 10⁵ pfu,approximately 10⁶ pfu or approximately 10⁷ may be administered to thesubject. In certain embodiments, wherein the non-human subject isadministered an antigen derived or obtained from an Influenza virus,approximately 1 mg/kg, approximately 1.5 mg/kg, approximately 2 mg/kg,approximately 3 mg/kg or approximately 4 mg/kg may be administered tothe subject. In certain embodiments, wherein the non-human subject isadministered an antigen derived or obtained from an Influenza virus,about 0.1 μg to about 1,000 μg; about 0.1 μg to about 500 μg; about 0.1μg to about 250 μg; about 0.1 μg to about 100 μg; about 0.1 μg to about50 μg, about 0.1 μg to about 25 μg or about 0.1 μg to about 10 μg of theantigen may be administered to the subject. In certain embodiments,wherein the non-human subject is administered a nucleic acid encoding anantigen derived or obtained from an Influenza virus, about 10 ng to 1 g,100 ng to 100 mg, 1 μg to 10 mg or 30-300 μg of the nucleic acid may beadministered to the subject.

5.1.1 Influenza Viruses

In certain embodiments, the Influenza viruses administered to anon-human subject are inactivated. Techniques known to one of skill inthe art may be used to inactivate viruses. Common methods use formalin,heat, or detergent for inactivation. See, e.g., U.S. Pat. No. 6,635,246,which is herein incorporated by reference in its entirety. Other methodsinclude those described in U.S. Pat. Nos. 5,891,705; 5,106,619 and4,693,981, which are incorporated herein by reference in theirentireties.

In certain embodiments, the Influenza viruses administered to anon-human subject are attenuated (e.g., a live Influenza virus that hasbeen attenuated). In specific embodiments, attenuation of Influenzavirus is desired such that the virus remains, at least partially,infectious and can replicate in vivo, but only generate low titersresulting in subclinical levels of infection that are non-pathogenic.Such attenuated viruses are especially suited for embodiments describedherein wherein the virus or an immunogenic composition thereof isadministered to a non-human subject to induce an immune response.Attenuation of the Influenza virus can be accomplished according to anymethod known in the art, such as, e.g., selecting viral mutantsgenerated by chemical mutagenesis, mutation of the genome by geneticengineering, selecting reassortant viruses that contain segments withattenuated function, or selecting for conditional virus mutants (e.g.,cold-adapted viruses). Alternatively, naturally occurring attenuatedInfluenza viruses may be used as Influenza virus backbones for theInfluenza virus vectors.

In some embodiments, an Influenza virus may be attenuated, at least inpart, by engineering the Influenza virus to express a mutated NS 1 genethat impairs the ability of the virus to antagonize the cellularinterferon (IFN) response. Examples of the types of mutations that canbe introduced into the Influenza virus NS 1 gene include deletions,substitutions, insertions and combinations thereof. One or moremutations can be introduced anywhere throughout the NS1 gene (e.g., theN-terminus, the C-terminus or somewhere in between) and/or theregulatory element of the NS1 gene. In one embodiment, an attenuatedInfluenza virus comprises a genome having a mutation in an Influenzavirus NS 1 gene resulting in a deletion consisting of 5, preferably 10,15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 75, 80, 85, 90, 95, 99, 100,105, 110, 115, 120, 125, 126, 130, 135, 140, 145, 150, 155, 160, 165,170 or 175 amino acid residues from the C-terminus of NS1, or a deletionof between 5-170, 25-170, 50-170, 100-170, 100-160, or 105-160 aminoacid residues from the C-terminus. In another embodiment, an attenuatedInfluenza virus comprises a genome having a mutation in an Influenzavirus NS1 gene such that it encodes an NS1 protein of amino acidresidues 1-130, amino acid residues 1-126, amino acid residues 1-120,amino acid residues 1-115, amino acid residues 1-110, amino acidresidues 1-100, amino acid residues 1-99, amino acid residues 1-95,amino acid residues 1-85, amino acid residues 1-83, amino acid residues1-80, amino acid residues 1-75, amino acid residues 1-73, amino acidresidues 1-70, amino acid residues 1-65, or amino acid residues 1-60,wherein the N-terminus amino acid is number 1. For examples of NS1mutations and Influenza viruses comprising a mutated NS1, see, e.g.,U.S. Pat. Nos. 6,468,544 and 6,669,943; and Li et al., 1999, J. Infect.Dis. 179:1132-1138, each of which is incorporated by reference herein inits entirety.

5.1.2 Expression of Influenza Virus Antigen

A nucleic acid encoding an antigen derived or obtained from an Influenzavirus may be administered to a non-human subject as part of a vector,such as, e.g., an expression vector. In addition, an antigen derived orobtained from an Influenza virus may be produced by transfecting a hostcell with a nucleic acid encoding such antigen, and such nucleic acidmay be part of a vector. In a specific embodiment, the vector is anexpression vector that is capable of directing the expression of anucleic acid encoding an antigen derived or obtained from an Influenzavirus. Non-limiting examples of expression vectors include, but are notlimited to, plasmids and viral vectors, such as replication defectiveretroviruses, adenoviruses, adeno-associated viruses, Newcastle diseasevirus, vaccinia virus and baculoviruses. Standard molecular biologytechniques may be used to introduce a nucleic acid encoding an antigenderived or obtained from an Influenza virus into an expression vector.

An expression vector comprises a nucleic acid encoding an antigenderived or obtained from an Influenza virus in a form suitable forexpression of the nucleic acid in a host cell or non-human subject. In aspecific embodiment, an expression vector includes one or moreregulatory sequences, selected on the basis of the host cells to be usedfor expression, which is operably linked to the nucleic acid to beexpressed. Within an expression vector, “operably linked” is intended tomean that a nucleic acid of interest is linked to the regulatorysequence(s) in a manner which allows for expression of the nucleic acid(e.g., in an in vitro transcription/translation system or in a host cellwhen the vector is introduced into the host cell). Regulatory sequencesinclude promoters, enhancers and other expression control elements(e.g., polyadenylation signals). Regulatory sequences include thosewhich direct constitutive expression of a nucleic acid in many types ofhost cells, those which direct expression of the nucleic acid only incertain host cells (e.g., tissue-specific regulatory sequences), andthose which direct the expression of the nucleic acid upon stimulationwith a particular agent (e.g., inducible regulatory sequences). It willbe appreciated by those skilled in the art that the design of theexpression vector can depend on such factors as, e.g., the choice of thehost cell to be transformed, the level of expression of protein desired,etc.

Expression vectors can be designed for expression of an antigen derivedor obtained from an Influenza virus using prokaryotic (e g., E. coli) oreukaryotic cells (e.g., insect cells (using baculovirus expressionvectors), yeast cells or mammalian cells). Examples of mammalian hostcells include, but are not limited to, Crucell Per.C6 cells, Vero cells,CHO cells, VERY cells, BHK cells, HeLa cells, COS cells, MDCK cells, 293cells, 3T3 cells or WI38 cells. In certain embodiments, the hosts cellsare myeloma cells, e.g., NS0 cells, 45.6 TG1.7 cells, AF-2 clone 9B5cells, AF-2 clone 9B5 cells, J558L cells, MOPC 315 cells, MPC-11 cells,NCI-H929 cells, NP cells, NS0/1 cells, P3 NS1 Ag4 cells, P3/NS1/1-Ag4-1cells, P3U1 cells, P3X63Ag8 cells, P3X63Ag8.653 cells, P3X63Ag8U.1cells, RPMI 8226 cells, Sp20-Ag14 cells, U266B1 cells, X63AG8.653 cells,Y3.Ag.1.2.3 cells, and YO cells. Non-limiting examples of insect cellsinclude Sf9, Sf21, Trichoplusia ni, Spodoptera frugiperda and Bombyxmori. In a particular embodiment, a mammalian cell culture system (e.g.,Chinese hamster ovary or baby hamster kidney cells) is used forexpression of an Influenza hemagglutinin stem domain polypeptide.

In some embodiments, a plant cell culture system is used for expressionof an antigen derived or obtained from an Influenza virus. See, e.g.,U.S. Pat. Nos. 7,504,560; 6,770,799; 6,551,820; 6,136,320; 6,034,298;5,914,935; 5,612,487; and 5,484,719, and U.S. patent applicationpublication Nos. 2009/0208477, 2009/0082548, 2009/0053762, 2008/0038232,2007/0275014 and 2006/0204487 for plant cells and methods for theproduction of proteins utilizing plant cell culture systems.

In certain embodiments, plants (e.g., plants of the genus Nicotiana) maybe engineered to express an antigen derived or obtained from anInfluenza virus. In specific embodiments, plants are engineered toexpress a an antigen derived or obtained from an Influenza virus via anagroinfiltration procedure using methods known in the art. For example,nucleic acids encoding a gene of interest, e.g., a gene encoding anantigen derived or obtained from an Influenza virus, are introduced intoa strain of Agrobacterium. Subsequently the strain is grown in a liquidculture and the resulting bacteria are washed and suspended into abuffer solution. The plants are then exposed (e.g., via injection orsubmersion) to the Agrobacterium that comprises the nucleic acidsencoding an antigen derived or obtained from an Influenza virus suchthat the Agrobacterium transforms the gene of interest to a portion ofthe plant cells. The antigen derived or obtained from an Influenza virusis then transiently expressed by the plant and can isolated usingmethods known in the art and described herein. (For specific examplessee Shoji et al., 2008, Vaccine, 26(23):2930-2934; and D'Aoust et al.,2008, J. Plant Biotechnology, 6(9):930-940). In a specific embodiment,the plant is a tobacco plant (i.e., Nicotiana tabacum). In anotherspecific embodiment, the plant is a relative of the tobacco plant (e.g.,Nicotiana benthamiana). In other embodiments, algae (e.g., Chlamydomonasreinhardtii) may be engineered to express an antigen derived or obtainedfrom an Influenza virus (see, e.g., Rasala et al., 2010, PlantBiotechnology Journal (Published online Mar. 7, 2010)).

An expression vector can be introduced into host cells via conventionaltransformation or transfection techniques. Such techniques include, butare not limited to, calcium phosphate or calcium chlorideco-precipitation, DEAE-dextran-mediated transfection, lipofection, andelectroporation. Suitable methods for transforming or transfecting hostcells can be found in Sambrook et al., 1989, Molecular Cloning—ALaboratory Manual, 2nd Edition, Cold Spring Harbor Press, New York, andother laboratory manuals. In certain embodiments, a host cell istransiently transfected with an expression vector containing a nucleicacid encoding an antigen derived or obtained from an Influenza virus. Inother embodiments, a host cell is stably transfected with an expressionvector containing a nucleic acid encoding an antigen derived or obtainedfrom an Influenza virus.

For stable transfection of mammalian cells, it is known that, dependingupon the expression vector and transfection technique used, only a smallfraction of cells may integrate the foreign DNA into their genome. Inorder to identify and select these integrants, a nucleic acid thatencodes a selectable marker (e.g., for resistance to antibiotics) isgenerally introduced into the host cells along with the nucleic acid ofinterest. Examples of selectable markers include those which conferresistance to drugs, such as G418, hygromycin and methotrexate. Cellsstably transfected with the introduced nucleic acid can be identified bydrug selection (e.g., cells that have incorporated the selectable markergene will survive, while the other cells die).

As an alternative to recombinant expression of an antigen derived orobtained from an Influenza virus using a host cell, an expression vectorcontaining a nucleic acid encoding an antigen derived or obtained froman Influenza virus can be transcribed and translated in vitro using,e.g., T7 promoter regulatory sequences and T7 polymerase. In a specificembodiment, a coupled transcription/translation system, such as PromegaTNT®, or a cell lysate or cell extract comprising the componentsnecessary for transcription and translation may be used to produce anantigen derived or obtained from an Influenza virus.

Once an antigen derived or obtained from an Influenza virus has beenproduced, it may be isolated or purified by any method known in the artfor isolation or purification of a protein, for example, bychromatography (e.g., ion exchange, affinity, particularly by affinityfor the specific antigen, by Protein A, and sizing columnchromatography), centrifugation, differential solubility, or by anyother standard technique for the isolation or purification of proteins.

5.2 Antibodies

Provided herein are monoclonal antibodies generated in accordance withthe methods described herein that bind to and neutralize antigenicallydistinct strains of Influenza virus. In a specific embodiment, providedherein are monoclonal antibodies generated in accordance with themethods described herein that bind to and neutralize antigenicallydistinct strains of the H3 subtype of the Influenza A virus as measuredby techniques known to one of skill in the art, e.g., ELISA or Westernblot for binding and a microneutralization assay, such as described inExample 6 infra.

In a specific embodiment, provided herein are monoclonal antibodiesgenerated in accordance with the methods described herein that bind tothe HA region of a certain group, cluster or subtype of Influenza virus,e.g., Group 2 Influenza virus or the H3 subtype of the Influenza Avirus. In certain embodiments, the monoclonal antibodies generated inaccordance with the methods described herein have a higher affinity fora certain group, cluster or subtype of Influenza virus (e.g., Group 2Influenza virus or the H3 subtype of the Influenza A virus) than toanother group or subtype of Influenza virus. In specific embodiments,the affinity of a monoclonal antibody generated in accordance with themethods described herein for a certain group, cluster or subtype ofInfluenza virus (e.g., Group 2 Influenza virus or the H3 subtype of theInfluenza A virus) is 1-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold,6-fold, 7-fold, 8-fold, 9-fold, 10-fold, greater than 10-fold, 1- to2-fold, 1- to 5-fold, 1- to 10-fold, 2- to 5-fold, 2- to 10-fold, 5- to10-fold, 10- to 15-fold, or 10- to 20-fold greater than the affinity ofthe monoclonal antibody to another group, cluster or subtype ofInfluenza virus. In specific embodiments, the affinity of a monoclonalantibody generated in accordance with the methods described herein for acertain group, cluster or subtype of Influenza virus (e.g., Group 2Influenza virus or the H3 subtype of the Influenza A virus) is 0.5 log,1 log, 1.5 log, 2 log, 2.5 log, 3 log, 3.5 log, or 4 log greater thanthe affinity of the monoclonal antibody to another group, cluster orsubtype of Influenza virus.

In a specific embodiment, the monoclonal antibodies selectively bind tohemagglutinin expressed by one, two, three or more strains of Influenzavirus relative to a non-Influenza virus hemagglutinin antigen asassessed by techniques known in the art, e.g., ELISA, Western blot, FACsor BIACore. In other words, the monoclonal antibodies bind tohemagglutinin expressed by one, two, three or more strains of Influenzavirus with a higher affinity than a non-Influenza virus hemagglutininantigen as assessed by techniques known in the art, e.g., ELISA, Westernblot, FACs or BIACore. In specific embodiments, the monoclonalantibodies bind to hemagglutinin expressed by one, two, three or morestrains of Influenza virus with a 1-fold, 1.5-fold, 2-fold, 3-fold,4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, greater than10-fold, 1- to 2-fold, 1- to 5-fold, 1- to 10-fold, 2- to 5-fold, 2- to10-fold, 5- to 10-fold, 10- to 15-fold, or 10- to 20-fold greateraffinity than that which they bind to a non-Influenza virushemagglutinin antigen. In specific embodiments, the monoclonalantibodies bind to hemagglutinin expressed by one, two, three or morestrains of Influenza virus with a 0.5 log, 1 log, 1.5 log, 2 log, 2.5log, 3 log, 3.5 log, or 4 log greater affinity than that which they bindto a non-Influenza virus hemagglutinin antigen.

In a specific embodiment, a monoclonal antibody generated in accordancewith the methods described herein is capable of binding to the HA2region of the hemagglutinin polypeptide of the Influenza virus strainA/Hong Kong/1/1968 (H3). In another specific embodiment, an antibodygenerated in accordance with a method described herein is capable ofbinding to the long alpha-helix of the HA2 region of, e.g., theInfluenza virus strain A/Hong Kong/1/1968 (H3). In a specificembodiment, an antibody generated in accordance with a method describedherein binds to the long alpha-helix of the hemagglutinin polypeptide ofthe Influenza virus strain A/Hong Kong/1/1968 (H3) (i.e., amino acids76-130, numbered according to the classic H3 subtype numbering system),i.e., the antibody binds an epitope within the following amino acidsequence: RIQDLEKYVEDTKIDLWSYNAELLVALENQHTIDLTDSEMNKLFEKTRRQLRENA (SEQID NO:125). In another specific embodiment, a monoclonal antibodygenerated in accordance with a method described herein binds to aminoacid residues within the range of 304 to 513, 330 to 513, 345 to 513,360 to 513, 375 to 513, 390 to 513, and/or 405-513 of the hemagglutininpolypeptide of the Influenza virus strain A/Hong Kong/1/1968 (H3). Inanother specific embodiment, a monoclonal antibody generated inaccordance with a method described herein is capable of binding to aminoacid residues within the range of 330 to 513, 345 to 513, 359 to 513,360 to 513, 375 to 513, 390 to 513, 384 to 439, 405 to 435, and/or 405to 513 of the hemagglutinin polypeptide of the Influenza virus strainA/Hong Kong/1/1968 (H3) (i.e., amino acids 1-184, 16-184, 30-184,31-184, 46-184, 61-184, 70-110, 76-106, and/or 76-184 of thehemagglutinin polypeptide numbered according to the classic H3 subtypenumbering system). In another specific embodiment, a monoclonal antibodygenerated in accordance with the methods described herein is capable ofbinding to an epitope in the hemagglutinin polypeptide of A/HongKong/1/1968 (H3) located within amino acids 405 to 513 of thehemagglutinin polypeptide, i.e., the antibody binds an epitope withinthe following amino acid sequence: RIQDLEKYVE DTKIDLWSYN AELLVALENQHTIDLTDSEM NKLFEKTRRQ LRENAEDMGN GCFKIYHKCD NACIESIRNG TYDHDVYRDEALNNRFQIKG VELKSGYKD (SEQ ID NO:1).

In another specific embodiment, a monoclonal antibody generated inaccordance with the methods described herein is capable of binding to anepitope in the hemagglutinin polypeptide of A/Hong Kong/1/1968 (H3)located within amino acids 76-106, numbered according to the classic H3subtype numbering system (see, Wilson I A, Skehel J J, Wiley DC (1981)Structure of the haemagglutinin membrane glycoprotein of influenza virusat 3 A resolution. Nature 289:366-373 for classic H3 subtype numberingsystem), i.e., the antibody binds an epitope within the following aminoacid sequence: RIQDLEKYVEDTKIDLWSYNAELLVALENQH (SEQ ID NO:124). Inanother specific embodiment, a monoclonal antibody generated inaccordance with the methods described herein is capable of binding to anepitope in the hemagglutinin polypeptide of A/Hong Kong/1/1968 (H3)located within amino acids 73-103, 73-104, 73-105, 73-106, 73-107,73-108, 73-109, 74-103, 74-104, 74-105, 74-106, 74-107, 74-108, 74-109,75-103, 75-104, 75-105, 75-106, 75-107, 75-108, 75-109, 76-103, 76-104,76-105, 76-107, 76-108, 76-109, 77-103, 77-104, 77-105, 77-106, 77-107,77-108, 77-109, 78-103, 78-104, 78-105, 78-106, 78-107, 78-108, 78-109,79-103, 79-104, 79-105, 79-106, 79-107, 79-108, or 79-109 numberedaccording to the classic H3 subtype numbering system.

In a specific embodiment, a monoclonal antibody provided herein is theantibody designated 7A7. In another embodiment, a monoclonal antibodyprovided herein is the antibody designated 12D1. In another embodiment,a monoclonal provided is the antibody designated 39A4. In anotherembodiment, a monoclonal provided is the antibody designated 66A6.Encompassed herein are antigen-binding fragments (e.g., Fab fragments,F(ab′) fragments, F(ab′)₂ fragments) of the antibody designated 7A7, theantibody designated 12D1, the antibody designated 39A4, and the antibodydesignated 66A6. Hybridomas that produce each of the 7A7, 12D1, and 39A4antibodies were deposited under provisions of the Budapest Treaty withthe American Type Culture Collection (ATCC, 10801 University Blvd.,Manassas, Va. 20110-2209) on May 22, 2009 under ATCC Accession Nos.PTA-10058, PTA-10059, and PTA 10060, respectively, and are hereinincorporated by reference. A hybridoma that produces the 66A6 antibodywas deposited under provisions of the Budapest Treaty with the AmericanType Culture Collection (ATCC, 10801 University Blvd., Manassas, Va.20110-2209) on May 25, 2010 under ATCC Accession Nos. PTA-______, and isherein incorporated by reference.

Provided herein are antibodies (such as monoclonal antibodies) thatcompete with the 7A7 antibody, 12D1 antibody, 39A4 antibody, or 66A6antibody for binding to a strain of the H3 subtype of Influenza A virusas determined using techniques known to one of skill in the art. In aspecific embodiment, an antibody competes with the antibody 7A7, 12D1,39A4, or 66A6 for binding to the Influenza A virus strain A/HongKong/1/1968 (H3) as determined using techniques known to one of skill inthe art. In another specific embodiment, an antibody competes with theantibody 7A7, 12D1, 39A4, or 66A6 for binding to the Influenza A strainA/Alabama/1/1981 (H3) as determined using techniques known to one ofskill in the art. In another specific embodiment, an antibody competeswith the antibody 7A7, 12D1, 39A4, or 66A6 for binding to the InfluenzaA virus strain A/Beijing/47/1992 (H3) as determined using techniquesknown to one of skill in the art. In another specific embodiment, anantibody competes with the antibody 7A7, 12D1, 39A4, or 66A6 for bindingto the Influenza A virus strain A/Wyoming/3/2003 (H3) as determinedusing techniques known to one of skill in the art. Competition assaysknown to one of skill in the art may be used to assess the competitionof an antibody with the antibody 7A7, 12D1, 39A4, or 66A6 for binding toa strain of the H3 subtype of Influenza virus. For example, animmunoassay (e.g., an ELISA) in competitive format may be used.

Provided herein are antibodies that bind to a strain of Influenza Avirus which comprise a variable light (VL) chain and/or a variable heavy(VH) chain of the antibody 7A7, 12D1, 39A4, or 66A6. In one embodiment,an antibody that binds to a strain of Influenza A virus comprises the VLchain or VH chain of the antibody 7A7, 12D1, 39A4, or 66A6. In anotherembodiment, an antibody that binds to a strain of Influenza A viruscomprises the VL chain of the antibody 7A7, 12D1, 39A4, or 66A6 and theVH chain of another antibody. In another embodiment, an antibody thatbinds to a strain of Influenza A virus comprises the VH chain of theantibody 7A7, 12D1, 39A4, or 66A6 and the VL chain of another antibody.In a specific embodiment, an antibody that binds to a strain of theInfluenza A virus comprises the VL chain of the antibody 7A7 and the VHchain of the antibody 12D1, 39A4, or 66A6; the VL chain of the antibody12D1 and the VH chain of the antibody 7A7, 39A4, or 66A6; the VL chainof the antibody 39A4 and the VH chain of the antibody 7A7, 12D1, or66A6; the VH chain of the antibody 66A6 and the VL chain of the antibody7A7, 12D1, or 39A4; or the VL chain of the antibody 66A6 and the VHchain of the antibody 7A7, 12D1, or 39A4. In specific embodiments, suchantibodies bind to a strain of the H3 subtype of Influenza A virus andin certain embodiments, such antibodies neutralize a strain of the H3subtype of Influenza A virus.

Provided herein are antibodies that bind to a strain of Influenza Avirus which comprise a VL domain and/or a VH domain of the antibody 7A7,12D1, 39A4, or 66A6. In one embodiment, an antibody that binds to astrain of Influenza A virus comprises the VL domain or VH domain of theantibody 7A7, 12D1, 39A4, or 66A6. In another embodiment, an antibodythat binds to a strain of Influenza A virus comprises the VL domain ofthe antibody 7A7, 12D1, 39A4, or 66A6 and the VH domain of anotherantibody. In another embodiment, an antibody that binds to a strain ofInfluenza A virus comprises the VH domain of the antibody 7A7, 12D1,39A4, or 66A6 and the VL domain of another antibody. In a specificembodiment, an antibody that binds to a strain of the Influenza A viruscomprises the VL domain of the antibody 7A7 and the VH domain of theantibody 12D1, 39A4, or 66A6; the VL domain of the antibody 12D1 and theVH domain of the antibody 7A7, 39A4, or 66A6; the VL domain of theantibody 39A4 and the VH domain of the antibody 7A7, 12D1, or 66A6; theVH domain of the antibody 66A6 and the VL domain of the antibody 7A7,12D1, or 39A4; or the VL domain of the antibody 66A6 and the VH domainof the antibody 7A7, 12D1, or 39A4. In specific embodiments, suchantibodies bind to a strain of the H3 subtype of Influenza A virus andin certain embodiments, such antibodies neutralize a strain of the H3subtype of Influenza A virus. A VH domain or VL domain refers to thevariable region of the variable heavy chain or variable light chain,respectively.

Provided herein are antibodies that bind to a strain of Influenza Avirus which comprise a VL chain of the antibody 7A7, 12D1, 39A4, or 66A6and a VH domain of the antibody 7A7, 12D1, 39A4, or 66A6, or VL domainof the antibody 7A7, 12D1, 39A4, or 66A6 and a VH chain of the antibody7A7, 12D1, 39A4, or 66A6. In one embodiment, an antibody that binds to astrain of Influenza A virus comprises the VL chain of the antibody 7A7,12D1, 39A4, or 66A6 and the VH domain of another antibody. In anotherembodiment, an antibody that binds to a strain of Influenza A viruscomprises the VL domain of the antibody 7A7, 12D1, 39A4, or 66A6 and theVH chain of another antibody. In a specific embodiment, an antibody thatbinds to a strain of the Influenza A virus comprises the VL chain of theantibody 7A7 and the VH domain of the antibody 12D1, 39A4, or 66A6; theVL domain of the antibody 7A7 and the VH chain of the antibody 12D1,39A4, or 66A6; the VL chain of the antibody 12D1 and the VH domain ofthe antibody 7A7, 39A4, or 66A6; the VL domain of the antibody 12D1 andthe VH chain of the antibody 7A7, 39A4, or 66A6; the VL chain of theantibody 39A4 and the VH domain of the antibody 7A7, 12D1, or 66A6; theVL domain of the antibody 39A4 and the VH chain of the antibody 7A7,12D1, or 66A6; the VL chain of the antibody 66A6 and the VH domain ofthe antibody 7A7, 12D1, or 39A4; or the VL domain of the antibody 66A6and the VH chain of the antibody 7A7, 12D1, or 39A4. In specificembodiments, such antibodies bind to a strain of the H3 subtype ofInfluenza A virus and in certain embodiments, such antibodies neutralizea strain of the H3 subtype of Influenza A virus.

Provided herein are antibodies that bind to a strain of Influenza Avirus comprising one, two or three complementarity determining regions(CDRs) of the variable heavy chain (VH CDRs) of the antibody 7A7, 12D1,39A4, or 66A6 and one, two or three CDRs of the variable light chain (VLCDRs) of the antibody 7A7, 12D1, 39A4, or 66A6. In certain embodiments,an antibody that binds to a strain of Influenza A virus, comprises (oralternatively, consists of) a VH CDR1 and a VL CDR1; a VH CDR1 and a VLCDR2; a VH CDR1 and a VL CDR3; a VH CDR2 and a VL CDR1; VH CDR2 and a VLCDR2; a VH CDR2 and a VL CDR3; a VH CDR3 and a VL CDR1; a VH CDR3 and aVL CDR2; a VH CDR3 and a VL CDR3; a VH1 CDR1, a VH CDR2 and a VL CDR1; aVH CDR1, a VH CDR2 and a VL CDR2; a VH CDR1, a VH CDR2 and a VL CDR3; aVH CDR2, a VH CDR3 and a VL CDR1; a VH CDR2, a VH CDR3 and a VL CDR2; aVH CDR2, a VH CDR3 and a VL CDR3; a VH CDR1, a VL CDR1 and a VL CDR2; aVH CDR1, a VL CDR1 and a VL CDR3; a VH CDR2, a VL CDR1 and a VL CDR2; aVH CDR2, a VL CDR1 and a VL CDR3; a VH CDR3, a VL CDR1 and a VL CDR2; aVH CDR3, a VL CDR1 and a VL CDR3; a VH CDR1, a VH CDR2, a VH CDR3 and aVL CDR1; a VH CDR1, a VH CDR2, a VH CDR3 and a VL CDR2; a VH CDR1, a VHCDR2, a VH CDR3 and a VL CDR3; a VH CDR1, a VH CDR2, a VL CDR1 and a VLCDR2; a VH CDR1, a VH CDR2, a VL CDR1 and a VL CDR3; a VH CDR1, a VHCDR3, a VL CDR1 and a VL CDR2; a VH CDR1, a VH CDR3, a VL CDR1 and a VLCDR3; a VH CDR2, a VH CDR3, a VL CDR1 and a VL CDR2; a VH CDR2, a VHCDR3, a VL CDR1 and a VL CDR3; a VH CDR2, a VH CDR3, a VL CDR2 and a VLCDR3; a VH CDR1, a VH CDR2, a VH CDR3, a VL CDR1 and a VL CDR2; a VHCDR1, a VH CDR2, a VH CDR3, a VL CDR1 and a VL CDR3; a VH CDR1, a VHCDR2, a VL CDR1, a VL CDR2, and a VL CDR3; a VH CDR1, a VH CDR3, a VLCDR1, a VL CDR2, and a VL CDR3; a VH CDR2, a VH CDR3, a VL CDR1, a VLCDR2, and a VL CDR3; a VH CDR1, VH CDR2, a VH CDR3, a VL CDR1, a VLCDR2, and a VL CDR3; or any combination thereof of the VH CDRs and VLCDRs of the antibodies 7A7, 12D1, 39A4, or 66A6. In specificembodiments, such antibodies bind to a strain of the H3 subtype ofInfluenza A virus and in certain embodiments, such antibodies neutralizea strain of the H3 subtype of Influenza A virus.

The sequence of the antibody 7A7, 12D1, 39A4, and/or 66A6 can bedetermined using standard techniques known to one skilled in the art andthe VH chain, VL chain, VH domain, VL domain, VH CDRs, and VL CDRs canbe determined using, e.g., the Kabat numbering system (such as the EUindex in Kabat).

The deduced nucleotide sequences of the VH and VL chains of the antibody7A7 are shown in FIG. 19. The deduced amino acid sequences of the VH andVL chains of the antibody 7A7 are shown in FIG. 20. The deducednucleotide sequences of the VH and VL chains of the antibody 12D1 areshown in FIG. 21. The deduced amino acid sequences of the VH and VLchains of the antibody 12D1 are shown in FIG. 22. The deduced nucleotidesequences of the VH and VL chains of the antibody 66A6 are shown in FIG.28. The deduced amino acid sequences of the VH and VL chains of theantibody 66A6 are shown in FIG. 29. In FIGS. 19, 20, 21, 22, 28, and 29,the framework and CDR regions corresponding to the nucleic acidsequences are shown in bold and underlined, respectively. One of skillin the art can readily determine the location of the framework regionsand CDRs in amino acid sequences using techniques known in the art(e.g., using the program provided at the following websitewww.expasy.ch/tools/dna.html).

The antibodies provided herein or generated in accordance with themethods provided herein include derivatives that are chemicallymodified, i.e., by the covalent attachment of any type of molecule tothe antibody. For example, but not by way of limitation, the antibodyderivatives include antibodies that have been chemically modified, e.g.,by glycosylation, acetylation, pegylation, phosphorylation, amidation,derivatization by known protecting/blocking groups, proteolyticcleavage, linkage to a cellular ligand or other protein, etc. Any ofnumerous chemical modifications may be carried out by known techniques,including, but not limited to specific chemical cleavage, acetylation,formylation, metabolic synthesis of tunicamycin, etc. Additionally, thederivative may contain one or more non-classical amino acids.

The antibodies provided herein or generated in accordance with themethods provided herein can comprise a framework region known to thoseof skill in the art (e.g., a human or non-human fragment). The frameworkregion may be naturally occurring or consensus framework regions (see,e.g., Sui et al., 2009, Nature Structural & Molecular Biology16:265-273).

Also provided herein are nucleic acids encoding the antibodies providedherein or generated in accordance with the methods provided herein. Insome embodiments, a nucleic acid molecule(s) encoding an antibodyprovided herein or generated in accordance with the methods providedherein is isolated. In other embodiments, a nucleic acid(s) encoding anantibody provided herein or generated in accordance with the methodsprovided herein is not isolated. In yet other embodiments, a nucleicacid(s) encoding an antibody provided herein or generated in accordancewith the methods provided herein is integrated, e.g., into chromosomalDNA or an expression vector. In a specific embodiment, a nucleic acid(s)provided herein encodes for the antibody 7A7, 12D1, 39A4, 66A6 or afragment thereof (in particular, an antigen-binding fragment thereof).In another specific embodiment, a nucleic acid(s) provided hereinencodes for an antibody that binds to Influenza virus HA, wherein theantibody comprises the VH domain of the antibody 7A7, 12D1, 39A4, or66A6. In another specific embodiment, a nucleic acid(s) provided hereinencodes for an antibody that binds to Influenza virus HA, wherein theantibody comprises the VL domain of the antibody 7A7, 12D1, 39A4, or66A6. In another specific embodiment, a nucleic acid(s) provided hereinencodes for an antibody that binds to Influenza virus HA, wherein theantibody comprises the VH and VL domain of the antibody 7A7, 12D1, 39A4,or 66A6. In another specific embodiment, a nucleic acid(s) providedherein encodes for an antibody that binds to Influenza virus HA, whereinthe antibody comprises 1, 2, or 3 VH CDRs and/or 1, 2, or 3 VL CDRs ofthe antibody 7A7, 12D1, 39A4, or 66A6. In certain embodiments, thenucleic acid encodes an antibody that not only binds to Influenza virusHA, but also neutralizes the Influenza virus.

The antibodies described herein or generated in accordance with themethods provided herein can be affinity matured using techniques knownto one of skill in the art. The monoclonal antibodies described hereinor generated in accordance with the methods provided herein can bechimerized using techniques known to one of skill in the art. A chimericantibody is a molecule in which different portions of the antibody arederived from different immunoglobulin molecules. Methods for producingchimeric antibodies are known in the art. See, e.g., Morrison, 1985,Science 229:1202; Oi et al., 1986, BioTechniques 4:214; Gillies et al.,1989, J. Immunol. Methods 125:191-202; and U.S. Pat. Nos. 5,807,715,4,816,567, 4,816,397, and 6,331,415, which are incorporated herein byreference in their entirety.

The monoclonal antibodies described herein or generated in accordancewith the methods provided herein can be humanized. A humanized antibodyis an antibody which is capable of binding to a predetermined antigenand which comprises a framework region having substantially the aminoacid sequence of a human immunoglobulin and a CDR having substantiallythe amino acid sequence of a non-human immunoglobulin. A humanizedantibody comprises substantially all of at least one, and typically two,variable domains (Fab, Fab′, F(ab′)₂, Fab, Fv) in which all orsubstantially all of the CDR regions correspond to those of a non humanimmunoglobulin (i.e., donor antibody) and all or substantially all ofthe framework regions are those of a human immunoglobulin consensussequence. Preferably, a humanized antibody also comprises at least aportion of an immunoglobulin constant region (Fc), typically that of ahuman immunoglobulin. Ordinarily, the antibody will contain both thelight chain as well as at least the variable domain of a heavy chain.The antibody also may include the CH1, hinge, CH2, CH3, and CH4 regionsof the heavy chain. The humanized antibody can be selected from anyclass of immunoglobulins, including IgM, IgG, IgD, IgA and IgE, and anyisotype, including IgG1, IgG2, IgG3 and IgG4. Usually the constantdomain is a complement fixing constant domain where it is desired thatthe humanized antibody exhibit cytotoxic activity, and the class istypically IgG1. Where such cytotoxic activity is not desirable, theconstant domain may be of the IgG2 class. Examples of VL and VH constantdomains that can be used in certain embodiments include, but are notlimited to, C-kappa and C-gamma-1 (nG1m) described in Johnson et al.(1997) J. Infect. Dis. 176, 1215-1224 and those described in U.S. Pat.No. 5,824,307. The humanized antibody may comprise sequences from morethan one class or isotype, and selecting particular constant domains tooptimize desired effector functions is within the ordinary skill in theart. The framework and CDR regions of a humanized antibody need notcorrespond precisely to the parental sequences, e.g., the donor CDR orthe consensus framework may be mutagenized by substitution, insertion ordeletion of at least one residue so that the CDR or framework residue atthat site does not correspond to either the consensus or the importantibody. Such mutations, however, will not be extensive. Usually, atleast 75% of the humanized antibody residues will correspond to those ofthe parental framework and CDR sequences, more often 90%, and mostpreferably greater than 95%. Humanized antibodies can be produced usingvariety of techniques known in the art, including but not limited to,CDR-grafting (European Patent No. EP 239,400; International publicationNo. WO 91/09967; and U.S. Pat. Nos. 5,225,539, 5,530,101, and5,585,089), veneering or resurfacing (European Patent Nos. EP 592,106and EP 519,596; Padlan, 1991, Molecular Immunology 28(4/5):489-498;Studnicka et al., 1994, Protein Engineering 7(6):805-814; and Roguska etal., 1994, PNAS 91:969-973), chain shuffling (U.S. Pat. No. 5,565,332),and techniques disclosed in, e.g., U.S. Pat. No. 6,407,213, U.S. Pat.No. 5,766,886, WO 9317105, Tan et al., J. Immunol. 169:1119 25 (2002),Caldas et al., Protein Eng. 13(5):353-60 (2000), Morea et al., Methods20(3):267 79 (2000), Baca et al., J. Biol. Chem. 272(16):10678-84(1997), Roguska et al., Protein Eng. 9(10):895 904 (1996), Couto et al.,Cancer Res. 55 (23 Supp):5973s-5977s (1995), Couto et al., Cancer Res.55(8):1717-22 (1995), Sandhu J S, Gene 150(2):409-10 (1994), andPedersen et al., J. Mol. Biol. 235(3):959-73 (1994). See also U.S.Patent Pub. No. US 2005/0042664 A1 (Feb. 24, 2005), which isincorporated by reference herein in its entirety. Often, frameworkresidues in the framework regions will be substituted with thecorresponding residue from the CDR donor antibody to alter, preferablyimprove, antigen binding. These framework substitutions are identifiedby methods well known in the art, e.g., by modeling of the interactionsof the CDR and framework residues to identify framework residuesimportant for antigen binding and sequence comparison to identifyunusual framework residues at particular positions. (See, e.g., Queen etal., U.S. Pat. No. 5,585,089; and Reichmann et al., 1988, Nature332:323, which are incorporated herein by reference in their entireties.

5.2.1 Antibodies with Increased Half-Lives

Provided herein are antibodies, wherein said antibodies are modified tohave an extended (or increased) half-life in vivo. In particular,provided herein are modified antibodies which have a half-life in asubject, preferably a mammal and most preferably a human, of from about3 days to about 180 days (or more), and in some embodiments greater than3 days, greater than 7 days, greater than 10 days, greater than 15 days,greater than 20 days, greater than 25 days, greater than 30 days,greater than 35 days, greater than 40 days, greater than 45 days,greater than 50 days, at least about 60 days, greater than 75 days,greater than 90 days, greater than 105 days, greater than 120 days,greater than 135 days, greater than 150 days, greater than 165 days, orgreater than 180 days.

In a specific embodiment, modified antibodies having an increasedhalf-life in vivo are generated by introducing one or more amino acidmodifications (i.e., substitutions, insertions or deletions) into an IgGconstant domain, or FcRn-binding fragment thereof (preferably a Fc orhinge-Fc domain fragment). See, e.g., International Publication Nos. WO02/060919; WO 98/23289; and WO 97/34631; and U.S. Pat. No. 6,277,375;each of which is incorporated herein by reference in its entirety. In aspecific embodiment, the modified antibodies may have one or more aminoacid modifications in the second constant CH2 domain (residues 231-340of human IgG1) and/or the third constant CH3 domain (residues 341-447 ofhuman IgG1), with numbering according to the Kabat numbering system(e.g., the EU index in Kabat).

In some embodiments, to prolong the in vivo serum circulation ofantibodies, inert polymer molecules such as high molecular weightpolyethyleneglycol (PEG) are attached to the antibodies with or withouta multifunctional linker either through site-specific conjugation of thePEG to the N- or C-terminus of the antibodies or via epsilon-aminogroups present on lysine residues. Linear or branched polymerderivatization that results in minimal loss of biological activity willbe used. The degree of conjugation can be closely monitored by SDS-PAGEand mass spectrometry to ensure proper conjugation of PEG molecules tothe antibodies. Unreacted PEG can be separated from antibody-PEGconjugates by size-exclusion or by ion-exchange chromatography.PEG-derivatized antibodies can be tested for binding activity as well asfor in vivo efficacy using methods well-known to those of skill in theart, for example, by immunoassays described herein.

In another embodiment, antibodies are conjugated to albumin in order tomake the antibody more stable in vivo or have a longer half-life invivo. The techniques are well-known in the art, see, e.g., InternationalPublication Nos. WO 93/15199, WO 93/15200, and WO 01/77137; and EuropeanPatent No. EP 413,622, all of which are incorporated herein byreference.

5.2.2 Antibody Conjugates

In some embodiments, antibodies are conjugated or recombinantly fused toa diagnostic, detectable or therapeutic agent or any other molecule.When in vivo half-life is desired to be increased, said antibodies canbe modified antibodies. The conjugated or recombinantly fused antibodiescan be useful, e.g., for monitoring or prognosing the onset,development, progression and/or severity of an Influenza virus diseaseas part of a clinical testing procedure, such as determining theefficacy of a particular therapy. Such diagnosis and detection can beaccomplished by coupling the antibody to detectable substancesincluding, but not limited to, various enzymes, such as, but not limitedto, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, oracetylcholinesterase; prosthetic groups, such as, but not limited to,streptavidin/biotin and avidin/biotin; fluorescent materials, such as,but not limited to, umbelliferone, fluorescein, fluoresceinisothiocynate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; luminescent materials, such as, but notlimited to, luminol; bioluminescent materials, such as but not limitedto, luciferase, luciferin, and aequorin; radioactive materials, such as,but not limited to, iodine (¹³¹I, ¹²⁵I, ¹²³I, and ¹²¹I,), carbon (¹⁴C),sulfur (³⁵S), tritium (³H), indium (¹¹⁵In, ¹¹³In, ¹¹²In, and ¹¹¹In,),technetium (⁹⁹Tc), thallium (²⁰¹Ti), gallium (⁶⁸Ga, ⁶⁷Ga), palladium(¹⁰³Pd), molybdenum (⁹⁹Mo), xenon (¹³³Xe), fluorine (¹⁸F), ¹⁵³Sm, ¹⁷⁷Lu,¹⁵⁹Gd, ¹⁴⁹Pm, ¹⁴⁰La, ¹⁷⁵Yb, ¹⁶⁶Ho, ⁹⁰Y, ⁴⁷Sc, ¹⁸⁶Re, ¹⁸⁷Re, ¹⁴²Pr,¹⁰⁵Rh, ⁹⁷Ru, ⁶⁸Ge, ⁵⁷Co, ⁶⁵Zn, ⁸⁵Sr, ³²P, ¹⁵³Gd, ¹⁶⁹Yb, ⁵¹Cr, ⁵⁴Mn,⁷⁵Se, ¹¹³Sn, and ¹¹⁷Sn; and positron emitting metals using variouspositron emission tomographies, and non-radioactive paramagnetic metalions.

Encompassed herein are antibodies recombinantly fused or chemicallyconjugated (including both covalent and non-covalent conjugations) to aheterologous protein or polypeptide (or fragment thereof, preferably toa polypeptide of about 10, about 20, about 30, about 40, about 50, about60, about 70, about 80, about 90 or about 100 amino acids) to generatefusion proteins. In particular, provided herein are fusion proteinscomprising an antigen-binding fragment of a monoclonal antibody (e.g., aFab fragment, Fd fragment, Fv fragment, F(ab)₂ fragment, a VH domain, aVH CDR, a VL domain or a VL CDR) and a heterologous protein,polypeptide, or peptide. In a specific embodiment, the heterologousprotein, polypeptide, or peptide that the antibody is fused to is usefulfor targeting the antibody to a particular cell type.

In one embodiment, a fusion protein provided herein comprises the 7A7,12D1, 39A4, or 66A6 antibody and a heterologous polypeptide. In anotherembodiment, a fusion protein provided herein comprises anantigen-binding fragment of the 7A7, 12D1, 39A4, or 66A6 antibody and aheterologous polypeptide. In another embodiment, a fusion proteinprovided herein comprises one, two, or more VH domains having the aminoacid sequence of any one of the VH domains of the 7A7, 12D1, 39A4, or66A6 antibody or one or more VL domains having the amino acid sequenceof any one of the VL domains of the 7A7, 12D1, 39A4, or 66A6 antibodyand a heterologous polypeptide. In another embodiment, a fusion proteinprovided herein comprises one, two, or more VH CDRs having the aminoacid sequence of any one of the VH CDRs of the 7A7, 12D1, 39A4, or 66A6antibody and a heterologous polypeptide. In another embodiment, a fusionprotein comprises one, two, or more VL CDRs having the amino acidsequence of any one of the VL CDRs of the 7A7, 12D1, 39A4, or 66A6antibody and a heterologous polypeptide. In another embodiment, a fusionprotein provided herein comprises at least one VH domain and at leastone VL domain of the 7A7, 12D1, 39A4, or 66A6 antibody and aheterologous polypeptide. In yet another embodiment, a fusion proteinprovided herein comprises at least one VH CDR and at least one VL CDR ofthe 7A7, 12D1, 39A4, or 66A6 antibody and a heterologous polypeptide. Incertain embodiments, the above-referenced antibodies comprise a modifiedIgG (e.g., IgG1) constant domain, or FcRn binding fragment thereof(e.g., the Fc domain or hinge-Fc domain), described herein.

Encompassed herein are uses of the antibodies conjugated orrecombinantly fused to a therapeutic moiety or drug moiety that modifiesa given biological response. Therapeutic moieties or drug moieties arenot to be construed as limited to classical chemical therapeutic agents.For example, the drug moiety may be a protein, peptide, or polypeptidepossessing a desired biological activity. Such proteins may include, forexample, β-interferon, γ-interferon, α-interferon, interleukin-2(“IL-2”), interleukin-4 (“IL-4”), interleukin-6 (“IL-6”), interleukin-7(“IL-7”), interleukin 9 (“IL-9”), interleukin-10 (“IL-10”),interleukin-12 (“IL-12”), interleukin-15 (“IL-15”), interleukin-18(“IL-18”), interleukin-23 (“IL-23”), granulocyte macrophage colonystimulating factor (“GM-CSF”), granulocyte colony stimulating factor(“G-CSF”)), a growth factor, or a defensin. The therapeutic moiety ordrug conjugated or recombinantly fused to an antibody should be chosento achieve the desired prophylactic or therapeutic effect(s). In certainembodiments, an antibody conjugate may be used for the prophylactic ortherapeutic uses described herein. In certain embodiments, the antibodyis a modified antibody. A clinician or other medical personnel shouldconsider the following when deciding on which therapeutic moiety or drugto conjugate or recombinantly fuse to an antibody: the nature of thedisease, the severity of the disease, and the condition of the subject.

Moreover, antibodies can be fused to marker sequences, such as a peptideto facilitate purification. In preferred embodiments, the marker aminoacid sequence is a hexa-histidine peptide (i.e., His-tag), such as thetag provided in a pQE vector (QIAGEN, Inc.), among others, many of whichare commercially available. As described in Gentz et al., 1989, Proc.Natl. Acad. Sci. USA 86:821-824, for instance, hexa-histidine providesfor convenient purification of the fusion protein. Other peptide tagsuseful for purification include, but are not limited to, thehemagglutinin (“HA”) tag, which corresponds to an epitope derived fromthe Influenza hemagglutinin protein (Wilson et al., 1984, Cell 37:767),and the “flag” tag.

Methods for fusing or conjugating therapeutic moieties (includingpolypeptides) to antibodies are well known, see, e.g., Amon et al.,“Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy”,in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp.243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., “Antibodies For DrugDelivery”, in Controlled Drug Delivery (2nd Ed.), Robinson et al.(eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, “AntibodyCarriers Of Cytotoxic Agents In Cancer Therapy: A Review”, in MonoclonalAntibodies 84: Biological And Clinical Applications, Pinchera et al.(eds.), pp. 475-506 (1985); “Analysis, Results, And Future ProspectiveOf The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, inMonoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al.(eds.), pp. 303-16 (Academic Press 1985), Thorpe et al., 1982, Immunol.Rev. 62:119-58; —C—U.S. Pat. Nos. 5,336,603, 5,622,929, 5,359,046,5,349,053, 5,447,851, 5,723,125, 5,783,181, 5,908,626, 5,844,095, and5,112,946; EP 307,434; EP 367,166; EP 394,827; PCT publications WO91/06570, WO 96/04388, WO 96/22024, WO 97/34631, and WO 99/04813;Ashkenazi et al., Proc. Natl. Acad. Sci. USA, 88: 10535-10539, 1991;Traunecker et al., Nature, 331:84-86, 1988; Zheng et al., J. Immunol.,154:5590-5600, 1995; Vil et al., Proc. Natl. Acad. Sci. USA,89:11337-11341, 1992; which are incorporated herein by reference intheir entireties.

In particular, fusion proteins may be generated, for example, throughthe techniques of gene-shuffling, motif-shuffling, exon-shuffling,and/or codon-shuffling (collectively referred to as “DNA shuffling”).DNA shuffling may be employed to alter the activities of the monoclonalantibodies described herein or generated in accordance with the methodsprovided herein (e.g., antibodies with higher affinities and lowerdissociation rates). See, generally, U.S. Pat. Nos. 5,605,793,5,811,238, 5,830,721, 5,834,252, and 5,837,458; Patten et al., 1997,Curr. Opinion Biotechnol. 8:724-33; Harayama, 1998, Trends Biotechnol.16(2):76-82; Hansson, et al., 1999, J. Mol. Biol. 287:265-76; andLorenzo and Blasco, 1998, Biotechniques 24(2):308-313 (each of thesepatents and publications are hereby incorporated by reference in itsentirety). Antibodies, or the encoded antibodies, may be altered bybeing subjected to random mutagenesis by error-prone PCR, randomnucleotide insertion or other methods prior to recombination. Apolynucleotide encoding a monoclonal antibody described herein orgenerated in accordance with the methods provided herein may berecombined with one or more components, motifs, sections, parts,domains, fragments, etc. of one or more heterologous molecules.

An antibody can also be conjugated to a second antibody to form anantibody heteroconjugate as described by Segal in U.S. Pat. No.4,676,980, which is incorporated herein by reference in its entirety.

An antibody can also linked directly or indirectly to one or moreantibodies to produce bispecific/multispecific antibodies.

An antibody can also be attached to solid supports, which areparticularly useful for immunoassays or purification of an antigen. Suchsolid supports include, but are not limited to, glass, cellulose,polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.

5.3 Production of Antibody

The antibodies described herein can be produced by any method known inthe art for the synthesis of antibodies, in particular, by chemicalsynthesis or preferably, by recombinant expression techniques. Themethods provided herein encompass, unless otherwise indicated,conventional techniques in molecular biology, microbiology, geneticanalysis, recombinant DNA, organic chemistry, biochemistry, PCR,oligonucleotide synthesis and modification, nucleic acid hybridization,and related fields within the skill of the art. These techniques aredescribed in the references cited herein and are fully explained in theliterature. See, e.g., Maniatis et al. (1982) Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory Press; Sambrook et al.(1989), Molecular Cloning: A Laboratory Manual, Second Edition, ColdSpring Harbor Laboratory Press; Sambrook et al. (2001) MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.; Ausubel et al., Current Protocols in MolecularBiology, John Wiley & Sons (1987 and annual updates); Current Protocolsin Immunology, John Wiley & Sons (1987 and annual updates) Gait (ed.)(1984) Oligonucleotide Synthesis: A Practical Approach, IRL Press;Eckstein (ed.) (1991) Oligonucleotides and Analogues: A PracticalApproach, IRL Press; Birren et al. (eds.) (1999) Genome Analysis: ALaboratory Manual, Cold Spring Harbor Laboratory Press.

Recombinant expression of an antibody requires construction of anexpression vector containing a polynucleotide that encodes the antibody.Once a polynucleotide encoding an antibody has been obtained, the vectorfor the production of the antibody molecule may be produced byrecombinant DNA technology using techniques well-known in the art. Thus,methods for preparing a protein by expressing a polynucleotidecontaining an antibody encoding nucleotide sequence are describedherein. Methods which are well known to those skilled in the art can beused to construct expression vectors containing antibody codingsequences and appropriate transcriptional and translational controlsignals. These methods include, for example, in vitro recombinant DNAtechniques, synthetic techniques, and in vivo genetic recombination.Thus, provided herein are replicable vectors comprising a nucleotidesequence encoding an antibody operably linked to a promoter. Suchvectors may include the nucleotide sequence encoding the constant regionof the antibody and the variable domain of the antibody may be clonedinto such a vector for expression of the entire heavy, the entire lightchain, or both the entire heavy and light chains.

The expression vector is transferred to a host cell by conventionaltechniques and the transfected cells are then cultured by conventionaltechniques to produce a monoclonal antibody described herein orgenerated in accordance with the methods provided herein. Thus, providedherein are host cells containing a polynucleotide encoding a monoclonalantibody described herein or generated in accordance with the methodsprovided herein or fragments thereof, or a heavy or light chain thereof,or fragment thereof, or a single chain monoclonal antibody describedherein or generated in accordance with the methods provided herein,operably linked to a heterologous promoter. In preferred embodiments forthe expression of double-chained antibodies, vectors encoding both theheavy and light chains may be co-expressed in the host cell forexpression of the entire immunoglobulin molecule, as detailed below.

In a specific embodiment, a host cell provided herein comprises anucleic acid encoding the antibody 7A7, 12D1, 39A4, or 66A6. In anotherspecific embodiment, a host cell provided herein comprises a nucleicacid encoding an antibody that binds to Influenza virus HA, the antibodycomprising the VH chain or VH domain and/or the VL chain or VL domain ofthe antibody 7A7, 12D1, 39A4, or 66A6. In another specific embodiment, ahost cell provided herein comprises a nucleic acid encoding an antibodythat binds to Influenza virus HA, the antibody comprising the 1, 2, or 3VH CDRs and/or 1, 2, or 3 VL CDRs of the antibody 7A7, 12D1, 39A4, or66A6. In specific embodiments, the antibody not only binds to Influenzavirus HA, but also neutralizes the Influenza virus.

A variety of host-expression vector systems may be utilized to expressan antibody (see, e.g., U.S. Pat. No. 5,807,715). Such host-expressionsystems represent vehicles by which the coding sequences of interest maybe produced and subsequently purified, but also represent cells whichmay, when transformed or transfected with the appropriate nucleotidecoding sequences, express an antibody in situ. These include but are notlimited to microorganisms such as bacteria (e.g., E. coli and B.subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA orcosmid DNA expression vectors containing antibody coding sequences;yeast (e.g., Saccharomyces Pichia) transformed with recombinant yeastexpression vectors containing antibody coding sequences; insect cellsystems infected with recombinant virus expression vectors (e.g.,baculovirus) containing antibody coding sequences; plant cell systems(including plant cell systems described in Section 5.3) infected withrecombinant virus expression vectors (e.g., cauliflower mosaic virus,CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmidexpression vectors (e.g., Ti plasmid) containing antibody codingsequences; or mammalian cell systems (e.g., COS, CHO, BHK, 293, NS0, and3T3 cells) harboring recombinant expression constructs containingpromoters derived from the genome of mammalian cells (e.g.,metallothionein promoter) or from mammalian viruses (e.g., theadenovirus late promoter; the vaccinia virus 7.5K promoter). Preferably,bacterial cells such as Escherichia coli, and more preferably,eukaryotic cells, especially for the expression of whole recombinantantibody molecule, are used for the expression of a recombinant antibodymolecule. For example, mammalian cells such as Chinese hamster ovarycells (CHO), in conjunction with a vector such as the major intermediateearly gene promoter element from human cytomegalovirus is an effectiveexpression system for antibodies (Foecking et al., 1986, Gene 45:101;and Cockett et al., 1990, Bio/Technology 8:2). In a specific embodiment,the expression of nucleotide sequences encoding the monoclonalantibodies described herein or generated in accordance with the methodsprovided herein is regulated by a constitutive promoter, induciblepromoter or tissue specific promoter.

In bacterial systems, a number of expression vectors may beadvantageously selected depending upon the use intended for the antibodymolecule being expressed. For example, when a large quantity of such anantibody is to be produced, for the generation of pharmaceuticalcompositions of an antibody molecule, vectors which direct theexpression of high levels of fusion protein products that are readilypurified may be desirable. Such vectors include, but are not limited to,the E. coli expression vector pUR278 (Ruther et al., 1983, EMBO12:1791), in which the antibody coding sequence may be ligatedindividually into the vector in frame with the lac Z coding region sothat a fusion protein is produced; pIN vectors (Inouye & Inouye, 1985,Nucleic Acids Res. 13:3101-3109; Van Heeke & Schuster, 1989, J. Biol.Chem. 24:5503-5509); and the like. pGEX vectors may also be used toexpress foreign polypeptides as fusion proteins with glutathione5-transferase (GST). In general, such fusion proteins are soluble andcan easily be purified from lysed cells by adsorption and binding tomatrix glutathione agarose beads followed by elution in the presence offree glutathione. The pGEX vectors are designed to include thrombin orfactor Xa protease cleavage sites so that the cloned target gene productcan be released from the GST moiety.

In an insect system, Autographa californica nuclear polyhedrosis virus(AcNPV) is used as a vector to express foreign genes. The virus grows inSpodoptera frugiperda cells. The antibody coding sequence may be clonedindividually into non-essential regions (for example the polyhedringene) of the virus and placed under control of an AcNPV promoter (forexample the polyhedrin promoter).

In mammalian host cells, a number of viral-based expression systems maybe utilized. In cases where an adenovirus is used as an expressionvector, the antibody coding sequence of interest may be ligated to anadenovirus transcription/translation control complex, e.g., the latepromoter and tripartite leader sequence. This chimeric gene may then beinserted in the adenovirus genome by in vitro or in vivo recombination.Insertion in a non-essential region of the viral genome (e.g., region E1or E3) will result in a recombinant virus that is viable and capable ofexpressing the antibody molecule in infected hosts (e.g., see Logan &Shenk, 1984, Proc. Natl. Acad. Sci. USA 8 1:355-359). Specificinitiation signals may also be required for efficient translation ofinserted antibody coding sequences. These signals include the ATGinitiation codon and adjacent sequences. Furthermore, the initiationcodon must be in phase with the reading frame of the desired codingsequence to ensure translation of the entire insert. These exogenoustranslational control signals and initiation codons can be of a varietyof origins, both natural and synthetic. The efficiency of expression maybe enhanced by the inclusion of appropriate transcription enhancerelements, transcription terminators, etc. (see, e.g., Bittner et al.,1987, Methods in Enzymol. 153:51-544).

In addition, a host cell strain may be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications (e.g.,glycosylation) and processing (e.g., cleavage) of protein products maybe important for the function of the protein. Different host cells havecharacteristic and specific mechanisms for the post-translationalprocessing and modification of proteins and gene products. Appropriatecell lines or host systems can be chosen to ensure the correctmodification and processing of the foreign protein expressed. To thisend, eukaryotic host cells which possess the cellular machinery forproper processing of the primary transcript, glycosylation, andphosphorylation of the gene product may be used. Such mammalian hostcells include but are not limited to CHO, VERY, BHK, Hela, COS, Vero,MDCK, 293, 3T3, W138, BT483, Hs578T, HTB2, BT2O and T47D, NS0 (a murinemyeloma cell line that does not endogenously produce any immunoglobulinchains), CRL7O3O and HsS78Bst cells.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines which stably expressthe antibody molecule may be engineered. Rather than using expressionvectors which contain viral origins of replication, host cells can betransformed with DNA controlled by appropriate expression controlelements (e.g., promoter, enhancer, sequences, transcriptionterminators, polyadenylation sites, etc.), and a selectable marker.Following the introduction of the foreign DNA, engineered cells may beallowed to grow for 1-2 days in an enriched media, and then are switchedto a selective media. The selectable marker in the recombinant plasmidconfers resistance to the selection and allows cells to stably integratethe plasmid into their chromosomes and grow to form foci which in turncan be cloned and expanded into cell lines. This method mayadvantageously be used to engineer cell lines which express the antibodymolecule. Such engineered cell lines may be particularly useful inscreening and evaluation of compositions that interact directly orindirectly with the antibody molecule. Methods commonly known in the artof recombinant DNA technology may be routinely applied to select thedesired recombinant clone, and such methods are described, for example,in Ausubel et al. (eds.), Current Protocols in Molecular Biology, JohnWiley & Sons, NY (1993); Kriegler, Gene Transfer and Expression, ALaboratory Manual, Stockton Press, NY (1990); and in Chapters 12 and 13,Dracopoli et al. (eds.), Current Protocols in Human Genetics, John Wiley& Sons, NY (1994); Colberre-Garapin et al., 1981, J. Mol. Biol. 150:1,which are incorporated by reference herein in their entireties.

The expression levels of an antibody molecule can be increased by vectoramplification (for a review, see Bebbington and Hentschel, The use ofvectors based on gene amplification for the expression of cloned genesin mammalian cells in DNA cloning, Vol. 3 (Academic Press, New York,1987)). When a marker in the vector system expressing antibody isamplifiable, increase in the level of inhibitor present in culture ofhost cell will increase the number of copies of the marker gene. Sincethe amplified region is associated with the antibody gene, production ofthe antibody will also increase (Crouse et al., 1983, Mol. Cell. Biol.3:257).

The host cell may be co-transfected with two expression vectors providedherein, the first vector encoding a heavy chain derived polypeptide andthe second vector encoding a light chain derived polypeptide. The twovectors may contain identical selectable markers which enable equalexpression of heavy and light chain polypeptides. Alternatively, asingle vector may be used which encodes, and is capable of expressing,both heavy and light chain polypeptides. In such situations, the lightchain should be placed before the heavy chain to avoid an excess oftoxic free heavy chain (Proudfoot, 1986, Nature 322:52; and Kohler,1980, Proc. Natl. Acad. Sci. USA 77:2197-2199). The coding sequences forthe heavy and light chains may comprise cDNA or genomic DNA.

Once an antibody has been produced by recombinant expression, it may bepurified by any method known in the art for purification of animmunoglobulin molecule, for example, by chromatography (e.g., ionexchange, affinity, particularly by affinity for the specific antigenafter Protein A, and sizing column chromatography), centrifugation,differential solubility, or by any other standard technique for thepurification of proteins. Further, the antibodies may be fused toheterologous polypeptide sequences described herein or otherwise knownin the art to facilitate purification.

In addition, human antibodies could be generated using the antibodiesdescribed herein. Completely human antibodies which recognize a selectedepitope can be generated using a technique referred to as “guidedselection.” In this approach a selected non-human monoclonal antibody,e.g., a mouse antibody, is used to guide the selection of a completelyhuman antibody recognizing the same epitope. (Jespers et al.,Bio/technology 12:899-903 (1988)).

5.4 Compositions

Provided herein are compositions comprising an antibody having thedesired degree of purity in a physiologically acceptable carrier,excipient or stabilizer (Remington's Pharmaceutical Sciences (1990) MackPublishing Co., Easton, Pa.). In a specific embodiment, the compositionscomprise an antibody conjugated to a moiety such as described in Section5.2.2. In certain embodiments, the compositions comprise an antibodythat has been modified to increase its half-life. Acceptable carriers,excipients, or stabilizers are nontoxic to recipients at the dosages andconcentrations employed, and include buffers such as phosphate, citrate,and other organic acids; antioxidants including ascorbic acid andmethionine; preservatives (such as octadecyldimethylbenzyl ammoniumchloride; hexamethonium chloride; benzalkonium chloride, benzethoniumchloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methylor propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; andm-cresol); low molecular weight (less than about 10 residues)polypeptides; proteins, such as serum albumin, gelatin, orimmunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;amino acids such as glycine, glutamine, asparagine, histidine, arginine,or lysine; monosaccharides, disaccharides, and other carbohydratesincluding glucose, mannose, or dextrins; chelating agents such as EDTA;sugars such as sucrose, mannitol, trehalose or sorbitol; salt-formingcounter-ions such as sodium; metal complexes (e.g., Zn-proteincomplexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ orpolyethylene glycol (PEG).

In a specific embodiment, pharmaceutical compositions comprise anantibody, and optionally one or more additional prophylactic ortherapeutic agents, in a pharmaceutically acceptable carrier. In aspecific embodiment, pharmaceutical compositions comprise an effectiveamount of an antibody, and optionally one or more additionalprophylactic of therapeutic agents, in a pharmaceutically acceptablecarrier. In some embodiments, the antibody is the only active ingredientincluded in the pharmaceutical composition. Pharmaceutical compositionsdescribed herein can be useful in the prevention or treatment ofInfluenza virus infection. Further, pharmaceutical compositionsdescribed herein can be useful in the prevention, treatment ormanagement of Influenza virus disease.

Pharmaceutically acceptable carriers used in parenteral preparationsinclude aqueous vehicles, nonaqueous vehicles, antimicrobial agents,isotonic agents, buffers, antioxidants, local anesthetics, suspendingand dispersing agents, emulsifying agents, sequestering or chelatingagents and other pharmaceutically acceptable substances. Examples ofaqueous vehicles include Sodium Chloride Injection, Ringers Injection,Isotonic Dextrose Injection, Sterile Water Injection, Dextrose andLactated Ringers Injection. Nonaqueous parenteral vehicles include fixedoils of vegetable origin, cottonseed oil, corn oil, sesame oil andpeanut oil. Antimicrobial agents in bacteriostatic or fungistaticconcentrations can be added to parenteral preparations packaged inmultiple-dose containers which include phenols or cresols, mercurials,benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoic acidesters, thimerosal, benzalkonium chloride and benzethonium chloride.Isotonic agents include sodium chloride and dextrose. Buffers includephosphate and citrate. Antioxidants include sodium bisulfate. Localanesthetics include procaine hydrochloride. Suspending and dispersingagents include sodium carboxymethylcelluose, hydroxypropylmethylcellulose and polyvinylpyrrolidone. Emulsifying agents includePolysorbate 80 (TWEEN® 80). A sequestering or chelating agent of metalions includes EDTA. Pharmaceutical carriers also include ethyl alcohol,polyethylene glycol and propylene glycol for water miscible vehicles;and sodium hydroxide, hydrochloric acid, citric acid or lactic acid forpH adjustment.

A pharmaceutical composition may be formulated for any route ofadministration to a subject. Specific examples of routes ofadministration include intranasal, oral, pulmonary, transdermal,intradermal, and parental. Parenteral administration, characterized byeither subcutaneous, intramuscular or intravenous injection, is alsocontemplated herein. Injectables can be prepared in conventional forms,either as liquid solutions or suspensions, solid forms suitable forsolution or suspension in liquid prior to injection, or as emulsions.The injectables, solutions and emulsions also contain one or moreexcipients. Suitable excipients are, for example, water, saline,dextrose, glycerol or ethanol. In addition, if desired, thepharmaceutical compositions to be administered can also contain minoramounts of non-toxic auxiliary substances such as wetting or emulsifyingagents, pH buffering agents, stabilizers, solubility enhancers, andother such agents, such as for example, sodium acetate, sorbitanmonolaurate, triethanolamine oleate and cyclodextrins.

Preparations for parenteral administration of an antibody includesterile solutions ready for injection, sterile dry soluble products,such as lyophilized powders, ready to be combined with a solvent justprior to use, including hypodermic tablets, sterile suspensions readyfor injection, sterile dry insoluble products ready to be combined witha vehicle just prior to use and sterile emulsions. The solutions may beeither aqueous or nonaqueous.

If administered intravenously, suitable carriers include physiologicalsaline or phosphate buffered saline (PBS), and solutions containingthickening and solubilizing agents, such as glucose, polyethyleneglycol, and polypropylene glycol and mixtures thereof.

Topical mixtures comprising an antibody are prepared as described forthe local and systemic administration. The resulting mixture can be asolution, suspension, emulsions or the like and can be formulated ascreams, gels, ointments, emulsions, solutions, elixirs, lotions,suspensions, tinctures, pastes, foams, aerosols, irrigations, sprays,suppositories, bandages, dermal patches or any other formulationssuitable for topical administration.

An antibody can be formulated as an aerosol for topical application,such as by inhalation (see, e.g., U.S. Pat. Nos. 4,044,126, 4,414,209,and 4,364,923, which describe aerosols for delivery of a steroid usefulfor treatment of inflammatory diseases, particularly asthma). Theseformulations for administration to the respiratory tract can be in theform of an aerosol or solution for a nebulizer, or as a microfine powderfor insufflations, alone or in combination with an inert carrier such aslactose. In such a case, the particles of the formulation will, in oneembodiment, have diameters of less than 50 microns, in one embodimentless than 10 microns.

An antibody can be formulated for local or topical application, such asfor topical application to the skin and mucous membranes, such as in theeye, in the form of gels, creams, and lotions and for application to theeye or for intracisternal or intraspinal application. Topicaladministration is contemplated for transdermal delivery and also foradministration to the eyes or mucosa, or for inhalation therapies. Nasalsolutions of the antibody alone or in combination with otherpharmaceutically acceptable excipients can also be administered.

Transdermal patches, including iontophoretic and electrophoreticdevices, are well known to those of skill in the art, and can be used toadminister an antibody. For example, such patches are disclosed in U.S.Pat. Nos. 6,267,983, 6,261,595, 6,256,533, 6,167,301, 6,024,975,6,010715, 5,985,317, 5,983,134, 5,948,433, and 5,860,957.

In certain embodiments, a pharmaceutical composition comprising anantibody is a lyophilized powder, which can be reconstituted foradministration as solutions, emulsions and other mixtures. It may alsobe reconstituted and formulated as solids or gels. The lyophilizedpowder is prepared by dissolving an antibody provided herein, or apharmaceutically acceptable derivative thereof, in a suitable solvent.In some embodiments, the lyophilized powder is sterile. The solvent maycontain an excipient which improves the stability or otherpharmacological component of the powder or reconstituted solution,prepared from the powder. Excipients that may be used include, but arenot limited to, dextrose, sorbitol, fructose, corn syrup, xylitol,glycerin, glucose, sucrose or other suitable agent. The solvent may alsocontain a buffer, such as citrate, sodium or potassium phosphate orother such buffer known to those of skill in the art at, in oneembodiment, about neutral pH. Subsequent sterile filtration of thesolution followed by lyophilization under standard conditions known tothose of skill in the art provides the desired formulation. In oneembodiment, the resulting solution will be apportioned into vials forlyophilization. Each vial will contain a single dosage or multipledosages of the compound. The lyophilized powder can be stored underappropriate conditions, such as at about 4° C. to room temperature.

Reconstitution of this lyophilized powder with water for injectionprovides a formulation for use in parenteral administration. Forreconstitution, the lyophilized powder is added to sterile water orother suitable carrier. The precise amount depends upon the selectedcompound. Such amount can be empirically determined.

An antibody can also, for example, be formulated in liposomes. Liposomescontaining the molecule of interest are prepared by methods known in theart, such as described in Epstein et al. (1985) Proc. Natl. Acad. Sci.USA 82:3688; Hwang et al. (1980) Proc. Natl. Acad. Sci. USA 77:4030; andU.S. Pat. Nos. 4,485,045 and 4,544,545. Liposomes with enhancedcirculation time are disclosed in U.S. Pat. No. 5,013,556. In oneembodiment, liposomal suspensions may also be suitable aspharmaceutically acceptable carriers. These can be prepared according tomethods known to those skilled in the art. For example, liposomeformulations can be prepared as described in U.S. Pat. No. 4,522,811.Briefly, liposomes such as multilamellar vesicles (MLV's) may be formedby drying down egg phosphatidyl choline and brain phosphatidyl serine(7:3 molar ratio) on the inside of a flask. A solution of a compoundcomprising monoclonal antibodies described herein or generated inaccordance with the methods provided herein provided herein in phosphatebuffered saline lacking divalent cations (PBS) is added and the flaskshaken until the lipid film is dispersed. The resulting vesicles arewashed to remove unencapsulated compound, pelleted by centrifugation,and then resuspended in PBS.

An antibody can also be entrapped in a microcapsule prepared, forexample, by coacervation techniques or by interfacial polymerization,for example, hydroxymethylcellulose or gelatin-microcapsule andpoly-(methylmethacylate) microcapsule, respectively, in colloidal drugdelivery systems (for example, liposomes, albumin microspheres,microemulsions, nano-particles and nanocapsules) or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences(1990) Mack Publishing Co., Easton, Pa.

Sustained-release preparations can also be prepared. Suitable examplesof sustained-release preparations include semipermeable matrices ofsolid hydrophobic polymers containing the antagonist, which matrices arein the form of shaped articles, e.g., films, or microcapsule. Examplesof sustained-release matrices include polyesters, hydrogels (forexample, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)),polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acidand ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the LUPRON DEPOT™(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid. Whilepolymers such as ethylene-vinyl acetate and lactic acid-glycolic acidenable release of molecules for over 100 days, certain hydrogels releaseproteins for shorter time periods. When encapsulated antibodies remainin the body for a long time, they may denature or aggregate as a resultof exposure to moisture at 37° C., resulting in a loss of biologicalactivity and possible changes in immunogenicity. Rational strategies canbe devised for stabilization depending on the mechanism involved. Forexample, if the aggregation mechanism is discovered to be intermolecularS—S bond formation through thio-disulfide interchange, stabilization maybe achieved by modifying sulfhydryl residues, lyophilizing from acidicsolutions, controlling moisture content, using appropriate additives,and developing specific polymer matrix compositions.

The compositions to be used for in vivo administration can be sterile.This is readily accomplished by filtration through, e.g., sterilefiltration membranes.

In a specific embodiment, nucleic acids comprising sequences encoding anantibody are administered to a subject by way of gene therapy. Genetherapy refers to therapy performed by the administration to a subjectof an expressed or expressible nucleic acid. Encompassed herein are anyof the methods for gene therapy available in the art. For general reviewof the methods of gene therapy, see Goldspiel et al., 1993, ClinicalPharmacy 12:488-505; Wu and Wu, 1991, Biotherapy 3:87-95; Tolstoshev,1993, Ann. Rev. Pharmacol. Toxicol. 32:573-596; Mulligan, 1993, Science260:926-932; and Morgan and Anderson, 1993, Ann. Rev. Biochem.62:191-217; May, 1993, TIBTECH 11(5):155-215. Methods commonly known inthe art of recombinant DNA technology which can be used are described inAusubel et al. (eds.), Current Protocols in Molecular Biology, JohnWiley & Sons, NY (1993); and Kriegler, Gene Transfer and Expression, ALaboratory Manual, Stockton Press, NY (1990).

5.5 Prophylactic and Therapeutic Uses

In one aspect, provided herein are methods of preventing, managing,and/or treating an Influenza virus disease in a subject by administeringneutralizing antibodies described herein. In a specific embodiment, amethod for preventing or treating an Influenza virus disease in asubject comprises administering to a subject an effective amount of aneutralizing antibody described herein, or a pharmaceutical compositionthereof. In another embodiment, a method for preventing, managing, ortreating an Influenza virus disease in a subject comprises administeringto a subject an effective amount of a neutralizing antibody describedherein, or a pharmaceutical composition thereof and another therapy. Inparticular embodiments, the neutralizing antibody is a monoclonalantibody. In a specific embodiment, the Influenza virus disease that isprevented, managed, or treated is caused by an Influenza virus that ischaracterized as a Group 2 Influenza virus. In another specificembodiment, the Influenza virus disease that is prevented, managed, ortreated is caused by an Influenza virus that is characterized as anInfluenza virus of the H3 subtype.

In one aspect, provided herein are methods of preventing or treating anInfluenza virus infection in a subject by administering neutralizingantibodies described herein. In a specific embodiment, a method forpreventing or treating an Influenza virus infection in a subjectcomprises administering to a subject an effective amount of aneutralizing antibody described herein, or a pharmaceutical compositionthereof. In another embodiment, a method for preventing or treating anInfluenza virus infection in a subject comprises administering to asubject an effective amount of a neutralizing antibody described herein,or a pharmaceutical composition thereof and another therapy. Inparticular embodiments, the neutralizing antibody is a monoclonalantibody.

In a specific embodiment, administration of an antibody(ies) prevents orinhibits Influenza virus from binding to its host cell receptor by atleast 99%, at least 95%, at least 90%, at least 85%, at least 80%, atleast 75%, at least 70%, at least 60%, at least 50%, at least 45%, atleast 40%, at least 45%, at least 35%, at least 30%, at least 25%, atleast 20%, or at least 10% relative to Influenza virus binding to itshost cell receptor in the absence of said antibody(ies) or in thepresence of a negative control in an assay known to one of skill in theart or described herein.

In a specific embodiment, administration of an antibody (ies) preventsor inhibits Influenza virus-induced fusion by at least 99%, at least95%, at least 90%, at least 85%, at least 80%, at least 75%, at least70%, at least 60%, at least 50%, at least 45%, at least 40%, at least45%, at least 35%, at least 30%, at least 25%, at least 20%, or at least10% relative to Influenza virus-induced fusion in the absence of saidantibody(ies) or in the presence of a negative control in an assay knownto one of skill in the art or described herein.

In a specific embodiment, administration of an antibody(ies) prevents orinhibits Influenza virus-induced fusion after viral attachment to cellsby at least 99%, at least 95%, at least 90%, at least 85%, at least 80%,at least 75%, at least 70%, at least 60%, at least 50%, at least 45%, atleast 40%, at least 45%, at least 35%, at least 30%, at least 25%, atleast 20%, or at least 10% relative to Influenza virus-induced fusionafter viral attachment to cells in the absence of said antibody(ies) orin the presence of a negative control in an assay known to one of skillin the art or described herein.

In a specific embodiment, administration of an antibody(ies) inhibits orreduces Influenza virus replication by at least 99%, at least 95%, atleast 90%, at least 85%, at least 80%, at least 75%, at least 70%, atleast 60%, at least 50%, at least 45%, at least 40%, at least 45%, atleast 35%, at least 30%, at least 25%, at least 20%, or at least 10%relative to replication of Influenza virus in the absence of saidantibody(ies) or in the presence of a negative control in an assay knownto one of skill in the art or described herein. Inhibition of Influenzavirus replication can be determined by detecting the Influenza virustiter in a biological specimens from a subject using methods known inthe art (e.g., Northern blot analysis, RT-PCR, Western Blot analysis,etc.).

In a specific embodiment, administration of an antibody(ies) results inreduction of about 1-fold, about 1.5-fold, about 2-fold, about 3-fold,about 4-fold, about 5-fold, about 8-fold, about 10-fold, about 15-fold,about 20-fold, about 25-fold, about 30-fold, about 35-fold, about40-fold, about 45-fold, about 50-fold, about 55-fold, about 60-fold,about 65-fold, about 70-fold, about 75-fold, about 80-fold, about85-fold, about 90-fold, about 95-fold, about 100-fold, about 105 fold,about 110-fold, about 115-fold, about 120 fold, about 125-fold or higherin Influenza virus titer in the subject. The fold-reduction in Influenzavirus titer may be as compared to a negative control, as compared toanother treatment, or as compared to the titer in the patient prior toantibody administration.

In a specific embodiment, administration of an antibody(ies) results ina reduction of approximately 1 log or more, approximately 2 logs ormore, approximately 3 logs or more, approximately 4 logs or more,approximately 5 logs or more, approximately 6 logs or more,approximately 7 logs or more, approximately 8 logs or more,approximately 9 logs or more, approximately 10 logs or more, 1 to 5logs, 2 to 10 logs, 2 to 5 logs, or 2 to 10 logs in Influenza virustiter in the subject. The log-reduction in Influenza virus titer may beas compared to a negative control, as compared to another treatment, oras compared to the titer in the patient prior to antibodyadministration.

In a specific embodiment, administration of an antibody(ies) inhibits orreduces Influenza virus infection of a subject by at least 99%, at least95%, at least 90%, at least 85%, at least 80%, at least 75%, at least70%, at least 60%, at least 50%, at least 45%, at least 40%, at least45%, at least 35%, at least 30%, at least 25%, at least 20%, or at least10% relative to Influenza virus infection of a subject in the absence ofsaid antibody(ies) or in the presence of a negative control in an assayknown to one of skill in the art or described herein.

In a specific embodiment, administration of an antibody(ies) inhibits orreduces the spread of Influenza virus in a subject by at least 99%, atleast 95%, at least 90%, at least 85%, at least 80%, at least 75%, atleast 70%, at least 60%, at least 50%, at least 45%, at least 40%, atleast 45%, at least 35%, at least 30%, at least 25%, at least 20%, or atleast 10% relative to the spread of Influenza virus in a subject in theabsence of said an antibody(ies) or in the presence of a negativecontrol in an assay known to one of skill in the art or describedherein.

In a specific embodiment, administration of an antibody(ies) inhibits orreduces the spread of Influenza virus between a subject and at least oneother subject by at least 99%, at least 95%, at least 90%, at least 85%,at least 80%, at least 75%, at least 70%, at least 60%, at least 50%, atleast 45%, at least 40%, at least 45%, at least 35%, at least 30%, atleast 25%, at least 20%, or at least 10% relative to the spread ofInfluenza virus between a subject and at least one other subject in theabsence of said antibody(ies) or in the presence of a negative controlin an assay known to one of skill in the art or described herein.

In a specific embodiment, administration of an antibody(ies) reduces thenumber of and/or the frequency of symptoms of Influenza virus disease orinfection in a subject (exemplary symptoms of influenza virus diseaseinclude, but are not limited to, body aches (especially joints andthroat), fever, nausea, headaches, irritated eyes, fatigue, sore throat,reddened eyes or skin, and abdominal pain).

An antibody(ies) may be administered alone or in combination withanother/other type of therapy known in the art to reduce Influenza virusinfection, to reduce titers of Influenza virus in a subject, to reducethe spread of Influenza virus between subjects, to inhibit Influenzavirus replication, to inhibit Influenza virus-induced fusion, and/or toinhibit binding of Influenza virus to its host cell receptor.

One or more of the antibodies may be used locally or systemically in thebody as a prophylactic or therapeutic agent. The antibodies may also beadvantageously utilized in combination with other antibodies (e.g.,monoclonal or chimeric antibodies), or with lymphokines or hematopoieticgrowth factors (such as, e.g., IL-2, IL-3 and IL-7), which, for example,serve to increase the number or activity of effector cells whichinteract with the antibodies.

One or more antibodies may also be advantageously utilized incombination with one or more agents used to treat Influenza virusinfection such as, for example anti-viral agents. Specific anti-viralagents include: oseltamavir (Tamiflu®), zanamivir (Relenza®), nucleosideanalogs (e.g., zidovudine, acyclovir, gangcyclovir, vidarabine,idoxuridine, trifluridine, and ribavirin), foscarnet, amantadine,rimantadine (Flumadine®), saquinavir, indinavir, ritonavir,alpha-interferons and other interferons, AZT, Influenza virus vaccines(e.g., Fluarix®, FluMist®, Fluvirin®, and Fluzone®).

In some embodiments, an antibody acts synergistically with the one ormore other therapies. Generally, administration of products of a speciesorigin or species reactivity (in the case of antibodies) that is thesame species as that of the patient is preferred. Thus, in a preferredembodiment, human or humanized antibodies are administered to a humanpatient for treatment or prophylaxis of an Influenza virus infection ora disease associated therewith.

In one embodiment, provided herein are methods of prevention,management, treatment and/or amelioration of an Influenza virus disease,and/or a symptom relating thereto as alternatives to current therapies.In a specific embodiment, the current therapy has proven or may prove tobe too toxic (i.e., results in unacceptable or unbearable side effects)for the patient. In another embodiment, a monoclonal antibody describedherein or generated in accordance with the methods provided hereindecreases the side effects as compared to the current therapy. Inanother embodiment, the patient has proven refractory to a currenttherapy. In such embodiments, encompassed herein is the administrationof one or more monoclonal antibodies described herein or generated inaccordance with the methods provided herein without any otheranti-infection therapies.

Suitable regimens can be selected by one skilled in the art byconsidering such factors and by following, for example, dosages reportedin the literature and recommended in the Physician's Desk Reference(58^(th) ed., 2004). See Section 5.5.2 for exemplary dosage amounts andfrequencies of administration of the monoclonal antibodies describedherein or generated in accordance with the methods provided herein.

In accordance with the methods encompassed herein, a monoclonal antibodydescribed herein or generated in accordance with the methods providedherein may be used as any line of therapy, including, but not limitedto, a first, second, third, fourth and/or fifth line of therapy.Further, in accordance with the methods encompassed herein, a monoclonalantibody described herein or generated in accordance with the methodsprovided herein can be used before or after any adverse effects orintolerance of the therapies other than a monoclonal antibody describedherein or generated in accordance with the methods provided hereinoccurs. Encompassed herein are methods for administering one or more amonoclonal antibody described herein or generated in accordance with themethods provided herein to prevent the onset of an Influenza virusdisease and/or to treat or lessen the recurrence of an Influenza virusdisease.

In a specific embodiment, administration of an antibody(ies) reduces theincidence of hospitalization by at least 99%, at least 95%, at least90%, at least 85%, at least 80%, at least 75%, at least 70%, at least60%, at least 50%, at least 45%, at least 40%, at least 45%, at least35%, at least 30%, at least 25%, at least 20%, or at least 10% relativeto the incidence of hospitalization in the absence of administration ofsaid antibody(ies).

In a specific embodiment, administration of an antibody(ies) reducesmortality by at least 99%, at least 95%, at least 90%, at least 85%, atleast 80%, at least 75%, at least 70%, at least 60%, at least 50%, atleast 45%, at least 40%, at least 45%, at least 35%, at least 30%, atleast 25%, at least 20%, or at least 10% relative to the mortality inthe absence of administration of said antibody(ies).

Further encompassed herein are methods for preventing, managing,treating and/or ameliorating an Influenza virus disease and/or a symptomrelating thereto for which no other anti-viral therapy is available.

5.5.1 Patient Population

In one embodiment, a patient treated or prevented in accordance with themethods provided herein is a naïve subject, i.e., a subject that doesnot have a disease caused by Influenza virus infection or has not beenand is not currently infected with an Influenza virus infection. Inanother embodiment, a patient treated or prevented in accordance withthe methods provided herein is a subject that is at risk of acquiring anInfluenza virus infection. In another embodiment, a patient treated orprevented in accordance with the methods provided herein is a naïvesubject that is at risk of acquiring an Influenza virus infection. Inanother embodiment, a patient treated or prevented in accordance withthe methods provided herein is a patient suffering from or expected tosuffer from an Influenza virus disease. In another embodiment, a patienttreated or prevented in accordance with the methods provided herein is apatient diagnosed with an Influenza virus infection or a diseaseassociated therewith. In some embodiments, a patient treated orprevented in accordance with the methods provided herein is a patientinfected with an Influenza virus that does not manifest any symptoms ofInfluenza virus disease.

In a specific embodiment, a patient treated or prevented in accordancewith the methods provided herein is a subject that is at risk of aninfection with a Group 2 Influenza virus. In another specificembodiment, a patient treated or prevented in accordance with themethods provided herein is a naïve subject that is at risk of aninfection with a Group 2 Influenza virus. In another specificembodiment, a patient treated or prevented in accordance with themethods provided herein is a patient suffering from or expected tosuffer from an Influenza virus disease caused by a Group 2 Influenzavirus. In another specific embodiment, a patient treated or prevented inaccordance with the methods provided herein is a patient diagnosed witha Group 2 Influenza virus infection or a disease associated therewith.

In another embodiment, a patient treated or prevented in accordance withthe methods provided herein is a patient experiencing one or moresymptoms of Influenza virus disease. Symptoms of Influenza virus diseaseinclude, but are not limited to, body aches (especially joints andthroat), fever, nausea, headaches, irritated eyes, fatigue, sore throat,reddened eyes or skin, and abdominal pain. In another embodiment, apatient treated or prevented in accordance with the methods providedherein is a patient with Influenza virus disease who does not manifestsymptoms of the disease that are severe enough to requirehospitalization.

In another embodiment, a patient treated or prevented in accordance withthe methods provided herein is a patient infected with an Influenza Avirus, an Influenza B virus or Influenza C virus. In another embodiment,a patient treated or prevented in accordance with the methods providedherein is a patient infected with a particular subtype of Influenza Avirus. In another embodiment, a patient treated or prevented inaccordance with the methods provided herein is a patient infected with aGroup 2 Influenza virus. In another embodiment, a patient treated orprevented in accordance with the methods provided herein is a patientinfected with an Influenza virus characterized as an Influenza virus ofthe H3 subtype. In accordance with such embodiments, the patients thatare infected with the virus may manifest symptoms of Influenza virusdisease.

In some embodiments, a patient treated or prevented in accordance withthe methods provided herein is an animal. In certain embodiments, theanimal is a bird. In certain embodiments, the animal is a mammal, e.g.,a horse, swine, mouse, or primate, preferably a human.

In a specific embodiment, a patient treated or prevented in accordancewith the methods provided herein is a human. In certain embodiments, apatient treated or prevented in accordance with the methods providedherein is a human infant. In some embodiments, a patient treated orprevented in accordance with the methods provided herein is a humantoddler. In certain embodiments, a patient treated or prevented inaccordance with the methods provided herein is a human child. In otherembodiments, a patient treated or prevented in accordance with themethods provided herein is a human adult. In some embodiments, a patienttreated or prevented in accordance with the methods provided herein isan elderly human.

In certain embodiments, a patient treated or prevented in accordancewith the methods provided herein is patient that is pregnant. In anotherembodiment, a patient treated or prevented in accordance with themethods provided herein is a patient who may or will be pregnant duringthe Influenza season (e.g., November to April in the NorthernHemisphere).

In some embodiments, a patient treated or prevented in accordance withthe methods provided herein is any subject at increased risk ofInfluenza virus infection or disease resulting from Influenza virusinfection (e.g., an immunocompromised or immunodeficient individual). Insome embodiments, a patient treated or prevented in accordance with themethods provided herein is any subject in close contact with anindividual with increased risk of Influenza virus infection or diseaseresulting from Influenza virus infection (e.g., immunocompromised orimmunosuppressed individuals).

In some embodiments, a patient treated or prevented in accordance withthe methods provided herein is a subject affected by any condition thatincreases susceptibility to Influenza virus infection or complicationsor disease resulting from Influenza virus infection. In otherembodiments, a patient treated or prevented in accordance with themethods provided herein is a subject in which an Influenza virusinfection has the potential to increase complications of anothercondition that the individual is affected by, or for which they are atrisk. In particular embodiments, such conditions that increasesusceptibility to Influenza virus complications or for which Influenzavirus increases complications associated with the condition are, e.g.,conditions that affect the lung, such as cystic fibrosis, asthma,emphysema, or bacterial infections; cardiovascular disease; or diabetes.Other conditions that may increase Influenza virus complications includekidney disorders; blood disorders (including anemia or sickle celldisease); or weakened immune systems (including immunosuppression causedby medications, malignancies such as cancer, organ transplant, or HIVinfection).

In some embodiments, a patient treated or prevented in accordance withthe methods provided herein is a subject that resides in a group home,such as a nursing home or orphanage. In some embodiments, a patienttreated or prevented in accordance with the methods provided herein issubject that works in, or spends a significant amount of time in, agroup home, e.g., a nursing home or orphanage. In some embodiments, apatient treated or prevented in accordance with the methods providedherein is a health care worker (e.g., a doctor or nurse). In someembodiments, a patient treated or prevented in accordance with themethods provided herein resides in a dormitory (e.g., a collegedormitory). In some embodiments, a patient treated or prevented inaccordance with the methods provided herein is a member of the military.In some embodiments, a patient treated or prevented in accordance withthe methods provided herein is a child that attends school.

In some embodiments, a patient treated or prevented in accordance withthe methods provided herein is a subject at increased risk of developingcomplications from Influenza virus infection including: any individualwho can transmit Influenza viruses to those at high risk forcomplications, such as, e.g., members of households with high-riskindividuals, including households that will include infants younger than6 months, individuals coming into contact with infants less than 6months of age, or individuals who will come into contact withindividuals who live in nursing homes or other long-term carefacilities; individuals with long-term disorders of the lungs, heart, orcirculation; individuals with metabolic diseases (e.g., diabetes);individuals with kidney disorders; individuals with blood disorders(including anemia or sickle cell disease); individuals with weakenedimmune systems (including immunosuppression caused by medications,malignancies such as cancer, organ transplant, or HIV infection);children who receive long-term aspirin therapy (and therefore have ahigher chance of developing Reye syndrome if infected with Influenza).

In other embodiments, a patient treated or prevented in accordance withthe methods provided herein includes healthy individuals six months ofage or older, who: plan to travel to foreign countries and areas whereflu outbreaks may be occurring, such, e.g., as the tropics and theSouthern Hemisphere from April through September; travel as a part oflarge organized tourist groups that may include persons from areas ofthe world where Influenza viruses are circulating; attend school orcollege and reside in dormitories, or reside in institutional settings;or wish to reduce their risk of becoming ill with Influenza virusdisease.

In specific embodiments, a patient treated or prevented in accordancewith the methods provided herein is an individual who is susceptible toadverse reactions to conventional therapies. In other embodiments, thepatient may be a person who has proven refractory to therapies otherthan an antibody described herein or generated in accordance with themethods provided herein but are no longer on these therapies. In certainembodiments, a patient with an Influenza virus disease is refractory toa therapy when the infection has not significantly been eradicatedand/or the symptoms have not been significantly alleviated. Thedetermination of whether a patient is refractory can be made either invivo or in vitro by any method known in the art for assaying theeffectiveness of a therapy for infections, using art-accepted meaningsof “refractory” in such a context. In various embodiments, a patientwith an Influenza virus disease is refractory when viral replication hasnot decreased or has increased following therapy.

In certain embodiments, patients treated or prevented in accordance withthe methods provided herein are patients already being treated withantibiotics, anti-virals, anti-fungals, or other biologicaltherapy/immunotherapy. Among these patients are refractory patients,patients who are too young for conventional therapies, and patients withreoccurring Influenza virus disease or a symptom relating theretodespite treatment with existing therapies.

5.5.2 Route of Administration and Dosage

An antibody or composition described herein may be delivered to asubject by a variety of routes. These include, but are not limited to,intranasal, intratracheal, oral, intradermal, intramuscular,intraperitoneal, transdermal, intravenous, conjunctival and subcutaneousroutes. Pulmonary administration can also be employed, e.g., by use ofan inhaler or nebulizer, and formulation with an aerosolizing agent foruse as a spray.

The amount of an antibody or composition which will be effective in thetreatment and/or prevention of an Influenza virus infection or anInfluenza virus disease will depend on the nature of the disease, andcan be determined by standard clinical techniques.

The precise dose to be employed in a composition will also depend on theroute of administration, and the seriousness of the infection or diseasecaused by it, and should be decided according to the judgment of thepractitioner and each subject's circumstances. For example, effectivedoses may also vary depending upon means of administration, target site,physiological state of the patient (including age, body weight, health),whether the patient is human or an animal, other medicationsadministered, and whether treatment is prophylactic or therapeutic.Usually, the patient is a human but non-human mammals includingtransgenic mammals can also be treated. Treatment dosages are optimallytitrated to optimize safety and efficacy.

In certain embodiments, an in vitro assay is employed to help identifyoptimal dosage ranges. Effective doses may be extrapolated from doseresponse curves derived from in vitro or animal model test systems.

For passive immunization with an antibody, the dosage ranges from about0.0001 to 100 mg/kg, and more usually 0.01 to 5 mg/kg, of the patientbody weight. For example, dosages can be 1 mg/kg body weight, 10 mg/kgbody weight, or within the range of 1-10 mg/kg or in other words, 70 mgor 700 mg or within the range of 70-700 mg, respectively, for a 70 kgpatient. In some embodiments, the dosage administered to the patient isabout 3 mg/kg to about 60 mg/kg of the patient's body weight.Preferably, the dosage administered to a patient is between 0.025 mg/kgand 20 mg/kg of the patient's body weight, more preferably 1 mg/kg to 15mg/kg of the patient's body weight. Generally, human antibodies have alonger half-life within the human body than antibodies from otherspecies due to the immune response to the foreign polypeptides. Thus,lower dosages of human antibodies and less frequent administration isoften possible. Further, the dosage and frequency of administration ofthe antibodies described herein or generated in accordance with themethods provided herein may be reduced by enhancing uptake and tissuepenetration (e.g., into the nasal passages and/or lung) of theantibodies by modifications such as, for example, lipidation.

An exemplary treatment regime entails administration once per every twoweeks or once a month or once every 3 to 6 months for a period of oneyear or over several years, or over several year-intervals. In somemethods, two or more antibodies with different binding specificities areadministered simultaneously to a subject. An antibody is usuallyadministered on multiple occasions. Intervals between single dosages canbe weekly, monthly, every 3 months, every 6 months or yearly. Intervalscan also be irregular as indicated by measuring blood levels of antibodyto the Influenza virus antigen (e.g., hemagglutinin) in the patient.

In a specific embodiment, an antibody described herein or generated inaccordance with the methods provided herein, or a composition thereof isadministered once a month just prior to (e.g., within three months,within two months, within one month) or during the Influenza season. Inanother embodiment, an antibody described herein or generated inaccordance with the methods provided herein, or a composition thereof isadministered every two months just prior to or during the Influenzaseason. In another embodiment, an antibody described herein or generatedin accordance with the methods provided herein, or a composition thereofis administered every three months just prior to or during the Influenzaseason. In a specific embodiment, an antibody described herein orgenerated in accordance with the methods provided herein, or acomposition thereof is administered once just prior to or during theInfluenza season. In another specific embodiment, an antibody describedherein or generated in accordance with the methods provided herein, or acomposition thereof is administered twice, and most preferably once,during a Influenza season. In some embodiments, an antibody describedherein or generated in accordance with the methods provided herein, or acomposition thereof is administered just prior to the Influenza seasonand can optionally be administered once during the Influenza season. Insome embodiments, an antibody described herein or generated inaccordance with the methods provided herein, or a composition thereof isadministered every 24 hours for at least three days, at least four days,at least five days, at least six days up to one week just prior to orduring an Influenza season. In specific embodiments, the dailyadministration of the antibody or composition thereof occurs soon afterInfluenza virus infection is first recognized in a patient, but prior topresentation of clinically significant disease. The term “Influenzaseason” refers to the season when Influenza infection is most likely tooccur. Typically, the Influenza season in the northern hemispherecommences in November and lasts through April.

In some embodiments, the plasma level of an antibody described herein orgenerated in accordance with the methods provided herein in a patient ismeasured prior to administration of a subsequent dose of an antibodydescribed herein or generated in accordance with the methods providedherein, or a composition thereof. The plasma level of the antibody maybe considered in determining the eligibility of a patient to receive asubsequent dose of an antibody described herein or generated inaccordance with the methods provided herein. For example, a patient'splasma level of an antibody described herein or generated in accordancewith the methods provided herein may suggest not administering anantibody described herein or generated in accordance with the methodsprovided herein; alternatively, a patient's plasma level of an antibodydescribed herein or generated in accordance with the methods providedherein may suggest administering an antibody described herein orgenerated in accordance with the methods provided herein at a particulardosage, at a particular frequency, and/or for a certain period of time.

In certain embodiments, the route of administration for a dose of anantibody described herein or generated in accordance with the methodsprovided herein, or a composition thereof to a patient is intranasal,intramuscular, intravenous, or a combination thereof, but other routesdescribed herein are also acceptable. Each dose may or may not beadministered by an identical route of administration. In someembodiments, an antibody described herein or generated in accordancewith the methods provided herein, or composition thereof, may beadministered via multiple routes of administration simultaneously orsubsequently to other doses of the same or a different antibodydescribed herein or generated in accordance with the methods providedherein.

5.5.3 Combination Therapies

In various embodiments, an antibody described herein or generated inaccordance with the methods provided herein or a nucleic acid encodingsuch an antibody may be administered to a subject in combination withone or more other therapies (e.g., antiviral or immunomodulatorytherapies). In some embodiments, a pharmaceutical composition describedherein may be administered to a subject in combination with one or moretherapies. The one or more other therapies may be beneficial in thetreatment or prevention of an Influenza virus disease or may amelioratea condition associated with an Influenza virus disease.

In some embodiments, the one or more other therapies that are supportivemeasures, such as pain relievers, anti-fever medications, or therapiesthat alleviate or assist with breathing. Specific examples of supportivemeasures include humidification of the air by an ultrasonic nebulizer,aerolized racemic epinephrine, oral dexamethasone, intravenous fluids,intubation, fever reducers (e.g., ibuprofen, acetometaphin), andantibiotic and/or anti-fungal therapy (i.e., to prevent or treatsecondary bacterial and/or fungal infections).

In certain embodiments, the therapies are administered less than 5minutes apart, less than 30 minutes apart, 1 hour apart, at about 1 hourapart, at about 1 to about 2 hours apart, at about 2 hours to about 3hours apart, at about 3 hours to about 4 hours apart, at about 4 hoursto about 5 hours apart, at about 5 hours to about 6 hours apart, atabout 6 hours to about 7 hours apart, at about 7 hours to about 8 hoursapart, at about 8 hours to about 9 hours apart, at about 9 hours toabout 10 hours apart, at about 10 hours to about 11 hours apart, atabout 11 hours to about 12 hours apart, at about 12 hours to 18 hoursapart, 18 hours to 24 hours apart, 24 hours to 36 hours apart, 36 hoursto 48 hours apart, 48 hours to 52 hours apart, 52 hours to 60 hoursapart, 60 hours to 72 hours apart, 72 hours to 84 hours apart, 84 hoursto 96 hours apart, or 96 hours to 120 hours part. In specificembodiments, two or more therapies are administered within the samepatent visit.

Any anti-viral agents well-known to one of skill in the art may be usedin combination with an antibody or pharmaceutical composition describedherein. Non-limiting examples of anti-viral agents include proteins,polypeptides, peptides, fusion proteins antibodies, nucleic acidmolecules, organic molecules, inorganic molecules, and small moleculesthat inhibit and/or reduce the attachment of a virus to its receptor,the internalization of a virus into a cell, the replication of a virus,or release of virus from a cell. In particular, anti-viral agentsinclude, but are not limited to, nucleoside analogs (e.g., zidovudine,acyclovir, gangcyclovir, vidarabine, idoxuridine, trifluridine, andribavirin), foscarnet, amantadine, rimantadine, saquinavir, indinavir,ritonavir, alpha-interferons and other interferons, AZT, zanamivir, andoseltamivir. Other anti-viral agents include Influenza virus vaccines,e.g., Fluarix® (GlaxoSmithKline), FluMist® (MedImmune Vaccines),Fluvirin® (Chiron Corporation), Fluzone® (Aventis Pasteur), or thosedescribed in Section 5.6 infra.

In specific embodiments, the anti-viral agent is an immunomodulatoryagent that is specific for a viral antigen. In particular embodiments,the viral antigen is an Influenza virus polypeptide other than ahemagglutinin polypeptide. In other embodiments, the viral antigen is anInfluenza virus hemagglutinin polypeptide.

In a specific embodiment, one or more therapies that prevent or treatsecondary responses to a primary Influenza virus infection areadministered in combination with one or more antibodies described hereinor generated in accordance with the methods provided herein. Examples ofsecondary responses to a primary Influenza virus infection include, butare not limited to, asthma-like responsiveness to mucosal stimuli,elevated total respiratory resistance, increased susceptibility tosecondary viral, bacterial, and fungal infections, and development ofconditions such as, but not limited to, bronchiolitis, pneumonia, croup,and febrile bronchitis.

In a specific embodiment, one or more antibodies described herein orgenerated in accordance with the methods provided herein is used incombination with another antibody (e.g., an anti-Influenza virusmonoclonal antibody) or a set of other antibodies (e.g., a set ofanti-Influenza virus monoclonal antibodies) in order to enhance theprophylactic and/or therapeutic effect of the other antibody or set ofother antibodies.

In some embodiments, a combination therapy comprises the administrationof one or more antibodies described herein or generated in accordancewith the methods provided herein. In some embodiments, a combinationtherapy comprises administration of two or more antibodies describedherein or generated in accordance with the methods provided herein. In aspecific embodiment, a combination therapy comprises the administrationof the 7A7 antibody and one or more of the antibody 12D1, 39A4, or 66A6.In another specific embodiment, a combination therapy comprises theadministration of the 12D1 antibody and one or more of the antibody 7A7,39A4, or 66A6. In another specific embodiment, a combination therapycomprises the administration of the 39A4 antibody and one or more of theantibody 7A7, 12D1, or 66A6. In another specific embodiment, acombination therapy comprises the administration of the 66A6 antibodyand one or more of the antibody 7A7, 39A4, or 12D1.

5.6 Use of the Antibodies to Generate Vaccines

Provided herein are immunogenic compositions (e.g., vaccines) capable ofgenerating immune responses against a plurality of Influenza virusstrains. While not intending to be bound by any particular theory ofoperation, it is believed that the antibody binding regions within theInfluenza antigens bound by an antibody described herein or generated bya process described herein are useful for presenting one or morerelatively conserved antigenic regions to a host immune system in orderto generate an immune response that is capable of cross-reacting with aplurality of Influenza strains. Since the one or more antigenic regionsare well conserved across Influenza subtypes, such an immune responsemight cross-react with several subtypes of Influenza virus.

Advantageously, Influenza regions bound by an antibody described hereinor generated by a process described herein might be useful to generatean immune response against multiple Influenza strains because theypresent one or more epitopes in a relatively conserved region of anInfluenza virus antigen. As such, they might be used to generate a hostimmune response against multiple Influenza strains that carry therelatively conserved epitopes. Accordingly, the Influenza hemagglutininregions bound by an antibody described herein or generated by a processdescribed herein find use as antigens in compositions and vaccines. TheInfluenza hemagglutinin regions bound by an antibody described herein orgenerated by a process described herein might be useful for generating ahost immune response against any one, two, three, four, five, six,seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen orsixteen known Influenza A subtypes or a later identified Influenza Asubtype. The Influenza hemagglutinin regions bound by an antibodydescribed herein or generated by a process described herein might beuseful for generating a host immune response against any one, two,three, four, five, six, seven, eight, nine, ten, eleven, twelve,thirteen, fourteen, fifteen or sixteen known Influenza A strains or alater identified Influenza A strain. The Influenza regions bound by anantibody described herein or generated by a process described hereinmight also be useful for generating a host immune response against anyInfluenza B subtype now known or later identified. Additionally, theantibodies described herein or generated in accordance with the methodsdescribed herein can be utilized to identify Influenza hemagglutininregions that mediate a broadly-protective immune response againstInfluenza viruses.

The binding regions of an antibody can be identified using methods knownin the art, e.g., Western blot, and described herein (see Section 6.3).Once an antibody binding region has been identified, the specificepitopes bound by the antibodies can be identified using methods knownin the art, e.g., alanine scanning mutagenesis (see, e.g., Cunningham etal., “High-Resolution Epitope Mapping of hGH-Receptor Interactions byAlanine-Scanning Mutagenesis” Science 244:1081-1085).

Generally, the Influenza virus binding regions and epitopes providedherein are polypeptides that comprise or consist essentially of thebinding regions and epitopes of an Influenza hemagglutinin polypeptidebound by an antibody described herein or generated by a processdescribed herein.

The Influenza virus binding regions and epitopes provided herein can beprepared according to any technique deemed suitable to one of skill,including techniques described below. In certain embodiments, theInfluenza virus binding regions and epitopes provided herein areisolated. In certain embodiments, the Influenza virus binding regionsand epitopes provided herein comprise a carbohydrate moiety.

In a specific embodiment, the Influenza virus binding region comprisesthe long alpha-helix of the HA2 region of a hemagglutinin polypeptide ofan Influenza virus of the H3 subtype.

In a specific embodiment, an Influenza virus binding region comprisesthe long alpha-helix of the HA2 region of a hemagglutinin polypeptide ofan Influenza A virus, such as the Influenza A virus strain A/HongKong/1/1968 (H3).

In a specific embodiment, the Influenza virus binding region comprisesamino acid residues 304 to 513, 330 to 513, 345 to 513, 359 to 513, 360to 513, 375 to 513, 359 to 514, and/or 360 to 514 of the hemagglutininpolypeptide of an Influenza virus of the H3 subtype. In a specificembodiment, the Influenza virus binding region comprises amino acidresidues 330 to 513, 345 to 513, 359 to 513, 360 to 513, 375 to 513, 390to 513, 384 to 439, 405 to 435, and/or 405 to 513 of the hemagglutininpolypeptide of the Influenza virus strain A/Hong Kong/1/1968 (H3) (i.e.,amino acids 1-184, 16-184, 30-184, 31-184, 46-184, 61-184, 70-110,76-106, and/or 76-184 of the hemagglutinin polypeptide numberedaccording to the classic H3 subtype numbering system). In a specificembodiment, the Influenza virus binding region comprises amino acidresidues 76-106 of the hemagglutinin polypeptide numbered according tothe classic H3 subtype numbering system. In another specific embodiment,the Influenza virus binding region comprises amino acid residues 73-103,73-104, 73-105, 73-106, 73-107, 73-108, 73-109, 74-103, 74-104, 74-105,74-106, 74-107, 74-108, 74-109, 75-103, 75-104, 75-105, 75-106, 75-107,75-108, 75-109, 76-103, 76-104, 76-105, 76-107, 76-108, 76-109, 77-103,77-104, 77-105, 77-106, 77-107, 77-108, 77-109, 78-103, 78-104, 78-105,78-106, 78-107, 78-108, 78-109, 79-103, 79-104, 79-105, 79-106, 79-107,79-108, or 79-109 numbered according to the classic H3 subtype numberingsystem.

In a specific embodiment, an Influenza virus binding region comprisesamino acid residues 304 to 513, 330 to 513, 345 to 513, 359 to 513, 360to 513, 375 to 513, 359 to 514, and/or 360 to 514 of the hemagglutininpolypeptide of the Influenza virus strain A/Hong Kong/1/1968 (H3). In aspecific embodiment, an Influenza virus binding region comprises aminoacid residues 330 to 513, 345 to 513, 359 to 513, 360 to 513, 375 to513, 390 to 513, 384 to 439, 405 to 435, and/or 405 to 513 of thehemagglutinin polypeptide of the Influenza virus strain A/HongKong/1/1968 (H3) (i.e., amino acids 1-184, 16-184, 30-184, 31-184,46-184, 61-184, 70-110, 76-106, and/or 76-184 of the hemagglutininpolypeptide numbered according to the classic H3 subtype numberingsystem). In another specific embodiment, an Influenza virus bindingregion provided herein comprises the following amino acid sequence:RIQDLEKYVE DTKIDLWSYN AELLVALENQ HTIDLTDSEM NKLFEKTRRQ LRENAEDMGNGCFKIYHKCD NACIESIRNG TYDHDVYRDE ALNNRFQIKG VELKSGYKD (SEQ ID NO:1). Inanother specific embodiment, an Influenza virus binding region providedherein comprises an amino acid sequence that is at least 99%, at least98%, and least 97%, at least 96%, at least 95%, at least 90%, at least85%, at least 80%, at least 75%, at least 70%, at least 65%, at least60%, at least 55% or at least 50% identical to the amino acid sequencein SEQ ID NO:1.

In another specific embodiment, an Influenza virus epitope providedherein comprises an epitope identified in SEQ ID NO:1 that is bound byan antibody described herein or generated in accordance with the methodsprovided herein. In another specific embodiment, an Influenza virusepitope provided herein comprises an amino acid sequence that is atleast 99%, at least 98%, and least 97%, at least 96%, at least 95%, atleast 90%, at least 85%, at least 80%, at least 75%, at least 70%, atleast 65%, at least 60%, at least 55% or at least 50% identical to anamino acid epitope identified in SEQ ID NO:1.

In certain embodiments, the binding region or epitope may be conjugatedor fused to a heterologous amino acid sequence. Such conjugated or fusedpolypeptides can be used in an immunogenic composition for the usesdescribed herein.

In certain embodiments, the binding region or epitope may becoupled/linked (e.g., via directly linked by a linker) to a carrierprotein, e.g., tetanus toxoid (CRM197—non-toxic diptheria toxoid pointmutant) or keyhole limpet hemocyanin (KLH).

Also provided herein are nucleic acids that encode an Influenza bindingregion and/or epitope provided herein. In a specific embodiment,provided herein is a nucleic acid that encodes an Influenza virusbinding region that comprises the long alpha-helix of the HA2 region ofa hemagglutinin polypeptide of an Influenza A virus, such as theInfluenza A virus strain A/Hong Kong/1/1968 (H3). In another specificembodiment, provided herein is a nucleic acid that encodes the longalpha-helix of the HA2 region of a hemagglutinin polypeptide of anInfluenza virus of the H3 subtype. In another specific embodiment,provided herein is a nucleic acid that encodes amino acid residues 304to 513, 330 to 513, 345 to 513, 359 to 513, 360 to 513, 375 to 513, 359to 514, and/or 360 to 514 of the hemagglutinin polypeptide of theInfluenza virus strain A/Hong Kong/1/1968 (H3). In another specificembodiment, provided herein is a nucleic acid that encodes amino acidresidues 330 to 513, 345 to 513, 359 to 513, 360 to 513, 375 to 513, 390to 513, 384 to 439, 405 to 435, and/or 405 to 513 of the hemagglutininpolypeptide of the Influenza virus strain A/Hong Kong/1/1968 (H3) (i.e.,amino acids 1-184, 16-184, 30-184, 31-184, 46-184, 61-184, 70-110,76-106, and/or 76-184 of the hemagglutinin polypeptide numberedaccording to the classic H3 subtype numbering system). In a specificembodiment, provided herein is a nucleic acid that encodes amino acidresidues 76-106 of the hemagglutinin polypeptide numbered according tothe classic H3 subtype numbering system. In another specific embodiment,provided herein is a nucleic acid that encodes amino acid residues73-103, 73-104, 73-105, 73-106, 73-107, 73-108, 73-109, 74-103, 74-104,74-105, 74-106, 74-107, 74-108, 74-109, 75-103, 75-104, 75-105, 75-106,75-107, 75-108, 75-109, 76-103, 76-104, 76-105, 76-107, 76-108, 76-109,77-103, 77-104, 77-105, 77-106, 77-107, 77-108, 77-109, 78-103, 78-104,78-105, 78-106, 78-107, 78-108, 78-109, 79-103, 79-104, 79-105, 79-106,79-107, 79-108, or 79-109 numbered according to the classic H3 subtypenumbering system. In a specific embodiment, provided herein is a nucleicacid that encodes SEQ ID NO:1 or an epitope identified within SEQ IDNO:1. Due to the degeneracy of the genetic code, any nucleic acid thatencodes SEQ ID NO:1 or an epitope identified within SEQ ID NO:1 isencompassed herein. In another specific embodiment, provided herein arenucleic acids that encode a binding region that is at least 99%, atleast 98%, and least 97%, at least 96%, at least 95%, at least 90%, atleast 85%, at least 80%, at least 75%, at least 70%, at least 65%, atleast 60%, at least 55% or at least 50% identical to the amino acidsequence in SEQ ID NO:1. In another specific embodiment, provided hereinare nucleic acids that encode an epitope that is at least 99%, at least98%, and least 97%, at least 96%, at least 95%, at least 90%, at least85%, at least 80%, at least 75%, at least 70%, at least 65%, at least60%, at least 55% or at least 50% identical to an epitope identified inSEQ ID NO:1. In some embodiments, the nucleic acids encompassed hereinare isolated. In accordance with the methods described herein, a nucleicacid that encodes an Influenza binding region and/or epitope providedherein can be administered to a patient to induce an immune response inthe patient.

Also provided herein are vectors, including expression vectors,containing nucleic acids that encode the binding regions and epitopesencompassed herein. In a specific embodiment, the vector is anexpression vector that is capable of directing the expression of anucleic acid that encodes a binding region and/or epitope encompassedherein.

Non-limiting examples of expression vectors include, but are not limitedto, plasmids and viral vectors, such as replication defectiveretroviruses, adenoviruses, adeno-associated viruses, Newcastle diseaseviruses, and baculoviruses. In certain embodiments, a binding region orepitope described herein is engineered into an influenza virus vector.In other embodiments, a binding region or epitope described herein isengineered into a non-Influenza virus. In certain embodiments, thebinding regions and epitopes encompassed herein can be incorporated intoviral-like particles or a virosome. Techniques known to one skilled inthe art may be used to produce expression vectors. In addition, anucleic acid encoding a binding region or epitope described herein, oran expression vector can be introduced into host cells using techniquesknown in the art (see, e.g., Sambrook et al., 1989, Molecular Cloning—ALaboratory Manual, 2nd Edition, Cold Spring Harbor Press, New York). Theexpression vector selected for expression of a binding region or epitopemay vary depending on the host cells chosen. The host cells selectedmight be prokaryotic (E. coli, Salmonella, Listeria, Shigella, etc.) oreukaryotic (e.g., mammalian cells, insect cells, yeast cells, or plantcells). The host cells may be engineered to stably or transientlyexpress a binding region or epitope (see, e.g., Section 5.1.2 forinformation regarding expression of antigens).

Accordingly, provided herein are methods for producing an Influenzavirus binding region and/or epitope. In one embodiment, the methodcomprises culturing a host cell containing a nucleic acid encoding thebinding region and/or epitope in a suitable medium such that the abinding region and/or epitope is produced. In some embodiments, themethod further comprises isolating the binding region and/or epitopefrom the medium or the host cell.

In one embodiment, an immunogenic composition comprises an Influenzavirus binding region and/or epitope provided herein, in an admixturewith a pharmaceutically acceptable carrier. In another embodiment, animmunogenic composition comprises a nucleic acid encoding an Influenzavirus binding region and/or epitope described herein, in an admixturewith a pharmaceutically acceptable carrier. In another embodiment, animmunogenic composition comprises an expression vector comprising anucleic acid encoding an Influenza virus binding region and/or epitopeprovided herein, in an admixture with a pharmaceutically acceptablecarrier. In another embodiment, an immunogenic composition comprises anInfluenza virus or non-Influenza virus containing an Influenza virusbinding region and/or epitope provided herein, in an admixture with apharmaceutically acceptable carrier. In another embodiment, animmunogenic composition comprises an Influenza virus or non-Influenzavirus having a genome engineered to express an Influenza virus bindingregion and/or epitope provided herein, in admixture with apharmaceutically acceptable carrier. In another embodiment, animmunogenic composition comprises a viral-like particle or virosomecontaining an Influenza virus binding region and/or epitope providedherein, in an admixture with a pharmaceutically acceptable carrier. Inanother embodiment, an immunogenic composition comprises a bacteriaexpressing or engineered to express an Influenza virus binding regionand/or epitope provided herein, in an admixture with a pharmaceuticallyacceptable carrier. In another embodiment, an immunogenic compositioncomprises cells stimulated with an Influenza virus binding region and/orepitope provided herein, in an admixture with a pharmaceuticallyacceptable carrier. In a specific embodiment, such compositions areformulated for the intended route of administration. Such compositionsmay include a pharmaceutically acceptable carrier or excipient.

In one embodiment, provided herein are subunit vaccines comprising anInfluenza virus binding region and/or epitope provided herein. In aspecific embodiment, a subunit vaccine comprises amino acid residues 304to 513, 330 to 513, 345 to 513, 359 to 513, 360 to 513, 375 to 513, 359to 514, and/or 360 to 514 of the hemagglutinin polypeptide of theInfluenza virus strain A/Hong Kong/1/1968 (H3). In a specificembodiment, a subunit vaccine comprises amino acid residues 330 to 513,345 to 513, 359 to 513, 360 to 513, 375 to 513, 390 to 513, 384 to 439,405 to 435, and/or 405 to 513 of the hemagglutinin polypeptide of theInfluenza virus strain A/Hong Kong/1/1968 (H3) (i.e., amino acids 1-184,16-184, 30-184, 31-184, 46-184, 61-184, 70-110, 76-106, and/or 76-184 ofthe hemagglutinin polypeptide numbered according to the classic H3subtype numbering system). In a specific embodiment, a subunit vaccinecomprises amino acid residues comprises amino acid residues 76-106 ofthe hemagglutinin polypeptide numbered according to the classic H3subtype numbering system. In another specific embodiment, a subunitvaccine comprises SEQ ID NO:1 or a nucleic acid encoding SEQ ID NO:1. Insome embodiments, the subunit vaccine further comprises one or moresurface glycoproteins (e.g., Influenza virus neuraminidase), othertargeting moieties, carrier proteins, or adjuvants.

In another embodiment, encompassed herein is a live virus engineered toexpress an Influenza virus binding region and/or epitope providedherein. In a specific embodiment, provided herein are immunogeniccompositions (e.g., vaccines) comprising live virus containing anInfluenza virus binding region and/or epitope provided herein. Inspecific embodiments, the Influenza virus binding region and/or epitopeprovided herein is membrane-bound. In other specific embodiments, theInfluenza virus binding region and/or epitope provided herein is notmembrane-bound, i.e., soluble. In particular embodiments, the live virusis an Influenza virus. In other embodiments, the live virus is anon-Influenza virus. In some embodiments, the live virus is attenuated.In some embodiments, an immunogenic composition comprises two, three,four or more live viruses containing or engineered to express two,three, four or more different Influenza virus binding regions and/orepitopes provided herein. In a specific embodiment, an immunogeniccomposition comprising live virus comprises amino acid residues 304 to513, 330 to 513, 345 to 513, 359 to 513, 360 to 513, 375 to 513, 359 to514, and/or 360 to 514 of the hemagglutinin polypeptide of the Influenzavirus strain A/Hong Kong/1/1968 (H3). In a specific embodiment, animmunogenic composition comprising live virus comprises amino acidresidues 330 to 513, 345 to 513, 359 to 513, 360 to 513, 375 to 513, 390to 513, 384 to 439, 405 to 435, and/or 405 to 513 of the hemagglutininpolypeptide of the Influenza virus strain A/Hong Kong/1/1968 (H3) (i.e.,amino acids 1-184, 16-184, 30-184, 31-184, 46-184, 61-184, 70-110,76-106, and/or 76-184 of the hemagglutinin polypeptide numberedaccording to the classic H3 subtype numbering system). In a specificembodiment, an immunogenic composition comprising live virus comprisesamino acid residues 76-106 of the hemagglutinin polypeptide numberedaccording to the classic H3 subtype numbering system. In anotherspecific embodiment, an immunogenic composition comprising live viruscomprises amino acid residues 73-103, 73-104, 73-105, 73-106, 73-107,73-108, 73-109, 74-103, 74-104, 74-105, 74-106, 74-107, 74-108, 74-109,75-103, 75-104, 75-105, 75-106, 75-107, 75-108, 75-109, 76-103, 76-104,76-105, 76-107, 76-108, 76-109, 77-103, 77-104, 77-105, 77-106, 77-107,77-108, 77-109, 78-103, 78-104, 78-105, 78-106, 78-107, 78-108, 78-109,79-103, 79-104, 79-105, 79-106, 79-107, 79-108, or 79-109 of thehemagglutinin polypeptide numbered according to the classic H3 subtypenumbering system. In a specific embodiment, an immunogenic compositioncomprising live virus comprises SEQ ID NO:1 or a nucleic acid encodingSEQ ID NO:1.

In another embodiment, provided herein are immunogenic compositions(e.g., vaccines) comprising an inactivated virus containing an Influenzavirus binding region and/or epitope provided herein. In specificembodiments, the Influenza virus binding region and/or epitope providedherein is membrane-bound. In particular embodiments, the inactivatedvirus is an Influenza virus. In other embodiments, the inactivated virusis a non-Influenza virus. In some embodiments, an immunogeniccomposition comprises two, three, four or more inactivated virusescontaining two, three, four or more different Influenza virus bindingregions and/or epitopes provided herein. In certain embodiments, theinactivated virus immunogenic compositions comprise one or moreadjuvants. In a specific embodiment, an immunogenic compositioncomprising an inactivated virus comprises amino acid residues 304 to513, 330 to 513, 345 to 513, 359 to 513, 360 to 513, 375 to 513, 359 to514, and/or 360 to 514 of the hemagglutinin polypeptide of the Influenzavirus strain A/Hong Kong/1/1968 (H3). In a specific embodiment, animmunogenic composition comprising an inactivated virus comprises aminoacid residues 330 to 513, 345 to 513, 359 to 513, 360 to 513, 375 to513, 390 to 513, 384 to 439, 405 to 435, and/or 405 to 513 of thehemagglutinin polypeptide of the Influenza virus strain A/HongKong/1/1968 (H3) (i.e., amino acids 1-184, 16-184, 30-184, 31-184,46-184, 61-184, 70-110, 76-106, and/or 76-184 of the hemagglutininpolypeptide numbered according to the classic H3 subtype numberingsystem). In a specific embodiment, an immunogenic composition comprisingan inactivated virus comprises amino acid residues 76-106 of thehemagglutinin polypeptide numbered according to the classic H3 subtypenumbering system. In another specific embodiment, an immunogeniccomposition comprising an inactivated virus comprises amino acidresidues 73-103, 73-104, 73-105, 73-106, 73-107, 73-108, 73-109, 74-103,74-104, 74-105, 74-106, 74-107, 74-108, 74-109, 75-103, 75-104, 75-105,75-106, 75-107, 75-108, 75-109, 76-103, 76-104, 76-105, 76-107, 76-108,76-109, 77-103, 77-104, 77-105, 77-106, 77-107, 77-108, 77-109, 78-103,78-104, 78-105, 78-106, 78-107, 78-108, 78-109, 79-103, 79-104, 79-105,79-106, 79-107, 79-108, or 79-109 of the hemagglutinin polypeptidenumbered according to the classic H3 subtype numbering system. In aspecific embodiment, an immunogenic composition comprising aninactivated virus comprises SEQ ID NO:1 or a nucleic acid encoding SEQID NO:1.

In another embodiment, an immunogenic composition comprising anInfluenza virus binding region and/or epitope provided herein is a splitvirus vaccine. In some embodiments, a split virus vaccine contains two,three, four or more different Influenza virus binding regions and/orepitopes provided herein. In certain embodiments, the Influenza virusbinding region and/or epitope provided herein is/was membrane-bound. Incertain embodiments, the split virus vaccines comprise one or moreadjuvants. In a specific embodiment, a split virus vaccine comprisesamino acid residues 330 to 513, 345 to 513, 359 to 513, 360 to 513, 375to 513, 390 to 513, 384 to 439, 405 to 435, and/or 405 to 513 of thehemagglutinin polypeptide of the Influenza virus strain A/HongKong/1/1968 (H3) (i.e., amino acids 1-184, 16-184, 30-184, 31-184,46-184, 61-184, 70-110, 76-106, and/or 76-184 of the hemagglutininpolypeptide numbered according to the classic H3 subtype numberingsystem). In a specific embodiment, a split virus vaccine comprises aminoacid residues 76-106 of the hemagglutinin polypeptide numbered accordingto the classic H3 subtype numbering system. In another specificembodiment, a split virus vaccine comprises amino acid residues 73-103,73-104, 73-105, 73-106, 73-107, 73-108, 73-109, 74-103, 74-104, 74-105,74-106, 74-107, 74-108, 74-109, 75-103, 75-104, 75-105, 75-106, 75-107,75-108, 75-109, 76-103, 76-104, 76-105, 76-107, 76-108, 76-109, 77-103,77-104, 77-105, 77-106, 77-107, 77-108, 77-109, 78-103, 78-104, 78-105,78-106, 78-107, 78-108, 78-109, 79-103, 79-104, 79-105, 79-106, 79-107,79-108, or 79-109 of the hemagglutinin polypeptide numbered according tothe classic H3 subtype numbering system. In another specific embodiment,a split virus vaccine comprises SEQ ID NO:1 or a nucleic acid encodingSEQ ID NO:1.

In certain embodiments, the binding regions and/or epitopes providedherein and/or immunogenic compositions comprising an Influenza virusbinding region and/or epitope provided herein can be used to induce animmune response to an Influenza A virus (e.g., any subtype or strain ofan Influenza A virus). In other embodiments, the binding regions and/orepitopes provided herein and/or immunogenic compositions comprising anInfluenza virus binding region and/or epitope provided herein can beused to induce an immune response to an Influenza virus characterized asa Group 2 Influenza virus. In other embodiments, the binding regionsand/or epitopes provided herein and/or immunogenic compositionscomprising an Influenza virus binding region and/or epitope providedherein can be used to induce an immune response to an Influenza virus ofthe H3 subtype.

In certain embodiments, the binding regions and/or epitopes providedherein and/or immunogenic compositions comprising an Influenza virusbinding region and/or epitope provided herein can be used to preventand/or treat an Influenza virus disease.

In certain embodiments, the binding regions and/or epitopes providedherein and/or immunogenic compositions comprising an Influenza virusbinding region and/or epitope provided herein can be used to preventand/or treat an Influenza virus infection.

In certain embodiments, the binding regions and/or epitopes providedherein and/or immunogenic compositions comprising an Influenza virusbinding region and/or epitope provided herein may be used in combinationwith another therapy (see, e.g., Section 5.5.3 for types of therapiesthat could be used in such a combination).

In certain embodiments, the binding regions and/or epitopes providedherein and/or immunogenic compositions comprising an Influenza virusbinding region and/or epitope provided herein can be can be administeredto patient by any route and maybe reference various routes. Theseinclude, but are not limited to, intranasal, intratracheal, oral,intradermal, intramuscular, intraperitoneal, transdermal, intravenous,conjunctival and subcutaneous routes. Pulmonary administration can alsobe employed, e.g., by use of an inhaler or nebulizer, and formulationwith an aerosolizing agent for use as a spray. Compositions can beformulated for the route of delivery.

Exemplary doses for nucleic acids encoding binding regions and/orepitopes provided herein range from about 10 ng to 1 g, 100 ng to 100mg, 1 μg to 10 mg, or 30-300 μg nucleic acid, e.g., DNA, per patient.

Exemplary doses for influenza the binding regions and/or epitopesprovided herein range from about 5 μg to 100 mg, 15 μg to 50 mg, 15 μgto 25 mg, 15 μg to 10 mg, 15 μg to 5 mg, 15 μg to 1 mg, 15 μg to 100 μg,15 μg to 75 μg, 5 μg to 50 μg, 10 μg to 50 μg, 15 μg to 45 μg, 20 μg to40 μg, or 25 to 35 μg per kilogram of the patient.

Doses for infectious viral vectors may vary from 10-100, or more,virions per dose. In some embodiments, suitable dosages of a virusvector are 10², 5×10², 10³, 5×10³, 10⁴, 5×10⁴, 10⁵, 5×10⁵, 10⁶, 5×10⁶,10⁷, 5×10⁷, 10⁸, 5×10⁸, 1×10⁹, 5×10⁹, 1×10¹⁰, 5×10¹⁰, 1×10¹¹, 5×10¹¹ or10¹² pfu, and can be administered to a subject once, twice, three ormore times with intervals as often as needed.

In one embodiment, an inactivated vaccine is formulated such that itcontains about 5 μg to about 50 μg, about 10 μg to about 50 μg, about 15μg to about 100 μg, about 15 μg to about 75 μg, about 15 μg to about 50μg, about 15 μg to about 30 μg, about 20 μg to about 50 μg, about 25 μgto about 40 μg, about 25 μg to about 35 μg of a binding region and/orepitope provided herein. Such a vaccine may contain a combination of oneor more different binding regions and/or epitopes provided herein.

Patients that can be administered the binding regions and/or epitopesprovided herein and/or immunogenic compositions comprising an Influenzavirus binding region and/or epitope provided herein include thoseidentified in Section 5.5.1.

5.7 Diagnostic Uses

The antibodies described herein or generated in accordance with themethods provided herein can be used for diagnostic purposes to detect anInfluenza virus as well as detect, diagnose, or monitor an Influenzavirus infection. In specific embodiments, the antibodies can be used todetermine whether a particular Influenza virus is present or aparticular Influenza virus subtype is present in a biological specimen(e.g., sputum, nasal drippings, other fluids, cells, or tissue samples).

Provided herein are methods for the detection of an Influenza virusinfection comprising: (a) assaying the expression of an Influenza virusantigen in a biological specimen (e.g., sputum, nasal drippings, cellsor tissue samples) from a subject using one or more of the antibodiesdescribed herein or generated in accordance with the methods providedherein; and (b) comparing the level of the Influenza virus antigen witha control level, e.g., levels in a biological specimen from a subjectnot infected with Influenza virus, wherein an increase in the assayedlevel of Influenza virus antigen compared to the control level of theInfluenza virus antigen is indicative of an Influenza virus infection.

In a specific embodiment, the subtype of the Influenza virus, e.g., theH3 subtype of Influenza A virus, can be detected in accordance with themethods for detecting an Influenza virus infection. According to thismethod, an antibody described herein or generated in accordance with themethods provided herein that is used in the assay is specificallyreactive to the subtype to be detected.

Provided herein is a diagnostic assay for diagnosing an Influenza virusinfection comprising: (a) assaying for the level of an Influenza virusantigen in a biological specimen from a subject using one or more of theantibodies described herein or generated in accordance with the methodsprovided herein; and (b) comparing the level of the Influenza virusantigen with a control level, e.g., levels in a biological specimen froma subject not infected with Influenza virus, wherein an increase in theassayed Influenza virus antigen level compared to the control level ofthe Influenza virus antigen is indicative of an Influenza virusinfection. A more definitive diagnosis of an Influenza virus infectionmay allow health professionals to employ preventative measures oraggressive treatment earlier thereby preventing the development orfurther progression of the Influenza virus infection.

Diagnosis of infection with a specific Influenza virus subtype (by useof subtype-specific antibodies) may allow the prescription of anti-viralmedications that are most appropriate for treatment of the particularsubtype.

Antibodies described herein or generated in accordance with the methodsprovided herein can be used to assay Influenza virus antigen levels in abiological sample using classical immunohistological methods asdescribed herein or as known to those of skill in the art (e.g., seeJalkanen et al., 1985, J. Cell. Biol. 101:976-985; and Jalkanen et al.,1987, J. Cell. Biol. 105:3087-3096). Antibody-based methods useful fordetecting protein expression include immunoassays, such as the enzymelinked immunosorbent assay (ELISA) and the radioimmunoassay (RIA). Anantibody described herein or generated in accordance with the methodsdescribed herein may be labeled with a detectable label or a secondaryantibody that binds to such an antibody may be labeled with a detectablelabel. Suitable antibody assay labels are known in the art and includeenzyme labels, such as, glucose oxidase; radioisotopes, such as iodine(¹²⁵I, ¹²¹I), carbon (¹⁴C), sulfur (³⁵S), tritium (³H), indium (¹²¹In),and technetium (⁹⁹Tc); luminescent labels, such as luminol; andfluorescent labels, such as fluorescein and rhodamine, and biotin.

Also provided herein is the detection and diagnosis of an Influenzavirus infection in a human. In one embodiment, diagnosis comprises: a)administering (for example, parenterally, intranasally, subcutaneously,or intraperitoneally) to a subject an effective amount of a labeledmonoclonal antibody described herein or generated in accordance with themethods provided herein; b) waiting for a time interval following theadministering for permitting the labeled antibody to preferentiallyconcentrate at sites in the subject (e.g., the nasal passages, lungs,mouth and ears) where the Influenza virus antigen is expressed (and forunbound labeled molecule to be cleared to background level); c)determining background level; and d) detecting the labeled antibody inthe subject, such that detection of labeled antibody above thebackground level indicates that the subject has an Influenza virusinfection or a symptom relating thereto. Background level can bedetermined by various methods, including comparing the amount of labeledmolecule detected to a standard value previously determined for aparticular system.

It will be understood in the art that the size of the subject and theimaging system used will determine the quantity of imaging moiety neededto produce diagnostic images. In the case of a radioisotope moiety, fora human subject, the quantity of radioactivity injected will normallyrange from about 5 to 20 millicuries of ⁹⁹Tc. The labeled antibody willthen preferentially accumulate at the location of cells which containthe specific protein. In vivo tumor imaging is described in S. W.Burchiel et al., “Immunopharmacokinetics of Radiolabeled Antibodies andTheir Fragments.” (Chapter 13 in Tumor Imaging: The RadiochemicalDetection of Cancer, S. W. Burchiel and B. A. Rhodes, eds., MassonPublishing Inc. (1982).

Depending on several variables, including the type of label used and themode of administration, the time interval following the administrationfor permitting the labeled antibody to preferentially concentrate atsites in the subject and for unbound labeled antibody to be cleared tobackground level is 6 to 48 hours, or 6 to 24 hours or 6 to 12 hours. Inanother embodiment the time interval following administration is 5 to 20days or 5 to 10 days.

In one embodiment, monitoring of an Influenza virus infection is carriedout by repeating the method for diagnosing the Influenza virusinfection, for example, one month after initial diagnosis, six monthsafter initial diagnosis, one year after initial diagnosis, etc.

Presence of the labeled molecule can be detected in the subject usingmethods known in the art for in vivo scanning. These methods depend uponthe type of label used. Skilled artisans will be able to determine theappropriate method for detecting a particular label. Methods and devicesthat may be used in the diagnostic methods provided herein include, butare not limited to, computed tomography (CT), whole body scan such asposition emission tomography (PET), magnetic resonance imaging (MRI),and sonography.

In a specific embodiment, the molecule is labeled with a radioisotopeand is detected in the patient using a radiation responsive surgicalinstrument (Thurston et al., U.S. Pat. No. 5,441,050). In anotherembodiment, the molecule is labeled with a fluorescent compound and isdetected in the patient using a fluorescence responsive scanninginstrument. In another embodiment, the molecule is labeled with apositron emitting metal and is detected in the patient using positronemission-tomography. In yet another embodiment, the molecule is labeledwith a paramagnetic label and is detected in a patient using magneticresonance imaging (MRI).

5.8 Biological Assays 5.8.1 Assays for Testing Antibody Activity

An antibody may be characterized in a variety of ways known to one ofskill in the art (e.g., ELISA, surface plasmon resonance display(BIAcore kinetic), Western blot, immunofluorescence, immunostainingand/or microneutralization assays). In some embodiments, an antibodyassayed for its ability to bind to an Influenza virus antigen (e.g., anhemagglutinin polypeptide), or an Influenza virus.

The specificity or selectivity of an antibody for an Influenza virusantigen (e.g., hemagglutinin polypeptide) or an Influenza virus andcross-reactivity with other antigens can be assessed by any method knownin the art. Immunoassays which can be used to analyze specific bindingand cross-reactivity include, but are not limited to, competitive andnon-competitive assay systems using techniques such as Western blots,radioimmunoassays, ELISA (enzyme linked immunosorbent assay), “sandwich”immunoassays, immunoprecipitation assays, precipitin reactions, geldiffusion precipitin reactions, immunodiffusion assays, agglutinationassays, complement-fixation assays, immunoradiometric assays,fluorescent immunoassays, protein A immunoassays, to name but a few.Such assays are routine and well known in the art (see, e.g., Ausubel etal., eds., 1994, Current Protocols in Molecular Biology, Vol. 1, JohnWiley & Sons, Inc., New York, which is incorporated by reference hereinin its entirety).

The binding affinity of an antibody to an Influenza virus antigen (e.g.,a hemagglutinin polypeptide) or an Influenza virus and the off-rate ofan antibody-antigen interaction can be determined by competitive bindingassays. One example of a competitive binding assay is a radioimmunoassaycomprising the incubation of labeled antigen (e.g., ³H or ¹²⁵I) with theantibody of interest in the presence of increasing amounts of unlabeledantigen, and the detection of the antibody bound to the labeled antigen.The affinity of the antibody for an Influenza virus antigen or anInfluenza virus and the binding off-rates can be determined from thedata by Scatchard plot analysis. Competition with a second antibody canalso be determined using radioimmunoassays. In this case, an Influenzavirus antigen or an Influenza virus is incubated with the test antibodyconjugated to a detectable labeled (e.g., ³H or ¹²⁵I) in the presence ofincreasing amounts of an unlabeled second antibody.

In some embodiments, surface plasmon resonance (e.g., BIAcore kinetic)analysis is used to determine the binding on and off rates of anantibody to an Influenza virus antigen (e.g., hemagglutininpolypeptide), or an Influenza virus. BIAcore kinetic analysis comprisesanalyzing the binding and dissociation of Influenza virus antigen fromchips with immobilized antibodies to an Influenza virus antigen on theirsurface. Briefly, a typical BIAcore kinetic study involves the injectionof 250 μL of an antibody reagent (mAb, Fab) at varying concentration inHBS buffer containing 0.005% Tween-20 over a sensor chip surface, ontowhich has been immobilized the Influenza virus hemagglutininpolypeptide. The flow rate is maintained constant at 75 μL/min.Dissociation data is collected for 15 min or longer as necessary.Following each injection/dissociation cycle, the bound antibody isremoved from the Influenza virus hemagglutinin polypeptide surface usingbrief, 1 min pulses of dilute acid, typically 10-100 mM HCl, thoughother regenerants are employed as the circumstances warrant. Morespecifically, for measurement of the rates of association, k_(on), anddissociation, k_(off), the polypeptide is directly immobilized onto thesensor chip surface through the use of standard amine couplingchemistries, namely the EDC/NHS method(EDC=N-diethylaminopropyl)-carbodiimide). Briefly, a 5-100 nM solutionof the polypeptide in 10 mM NaOAc, pH 4 or pH 5 is prepared and passedover the EDC/NHS-activated surface until approximately 30-50 RU's worthof polypeptide are immobilized. Following this, the unreacted activeesters are “capped” off with an injection of 1M Et-NH₂. A blank surface,containing no polypeptide, is prepared under identical immobilizationconditions for reference purposes. Once an appropriate surface has beenprepared, a suitable dilution series of each one of the antibodyreagents is prepared in HBS/Tween-20, and passed over both thepolypeptide and reference cell surfaces, which are connected in series.The range of antibody concentrations that are prepared varies, dependingon what the equilibrium binding constant, K_(D), is estimated to be. Asdescribed above, the bound antibody is removed after eachinjection/dissociation cycle using an appropriate regenerant.

The neutralizing activity of an antibody can be determined utilizing anyassay known to one skilled in the art. Antibodies can be assayed fortheir ability to inhibit the binding of an Influenza virus, or any othercomposition comprising Influenza virus antigen, such as a hemagglutininpolypeptide (e.g., a virus-like particle (VLP), liposome, or detergentextract), to its host cell receptor (i.e., sialic acid) using techniquesknown to those of skill in the art. For example, cells expressingInfluenza virus receptors can be contacted with a composition comprisingInfluenza virus antigen (e.g., a hemagglutinin polypeptide) in thepresence or absence of the antibody and the ability of the antibody toinhibit the antigen's binding can measured by, for example, flowcytometry or a scintillation assay. The composition comprising anInfluenza virus antigen or the antibody can be labeled with a detectablecompound such as a radioactive label (e.g., ³²P, ³⁵S, and ¹²⁵I) or afluorescent label (e.g., fluorescein isothiocyanate, rhodamine,phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde andfluorescamine) to enable detection of an interaction between thecomposition comprising an Influenza virus antigen and a cell receptor.Alternatively, the ability of an antibody to inhibit an Influenza virusantigen (e.g., a hemagglutinin polypeptide) from binding to its receptorcan be determined in cell-free assays. For example, a compositioncomprising an Influenza virus antigen (e.g., a hemagglutininpolypeptide) can be contacted with an antibody and the ability of theantibody to inhibit the composition comprising an Influenza virusantigen from binding to a cell receptor can be determined. In a specificembodiment, the antibody is immobilized on a solid support and thecomposition comprising an Influenza virus antigen is labeled with adetectable compound. Alternatively, a composition comprising anInfluenza virus antigen is immobilized on a solid support and theantibody is labeled with a detectable compound.

In a specific embodiment, the neutralizing activity of an antibody isassessed using a microneutralization assay as described in Section 6.1.4infra. In another specific embodiment, the neutralizing activity of anantibody is assessed using a plaque reduction assay as described inExample 6.1.5 infra.

In other embodiments, an antibody suitable for use in a method describedherein does not inhibit Influenza virus receptor binding, yet is stillfound to be neutralizing in an assay described herein. In someembodiments, an antibody suitable for use in accordance with a methoddescribed herein reduces or inhibits virus-host membrane fusion in anassay known in the art or described herein.

In one embodiment, virus-host membrane fusion is assayed in an in vitroassay using an Influenza virus containing a reporter and a host cellcapable of being infected with the virus. An antibody inhibits fusion ifreporter activity is inhibited or reduced compared to a negative control(e.g., reporter activity in the presence of a control antibody or in theabsence of antibody).

In one embodiment, virus-host membrane fusion is detected using a modelsystem of cell fusion. In an exemplary cell fusion assay, cells (e.g.,HeLa cells) are transfected with a plasmid encoding an Influenza virushemagglutinin polypeptide and contacted and exposed to a buffer thatallows the hemagglutinin polypeptide fusion function (e.g., pH 5.0buffer) in the presence of an antibody. An antibody is neutralizing ifit reduces or inhibits syncytia formation compared to a negative control(e.g., syncytia formation in the presence of a control antibody or inthe absence of antibody).

In a specific embodiment, virus-host membrane fusion is assayed using ared blood cell fusion assay as known in the art or described herein (seeSection 6.1.6 infra).

In other embodiments, virus-host membrane fusion is assayed using an invitro liposome-based assay. In an exemplary assay, the host cellreceptor is reconstituted into liposomes containing one half of areporter. Influenza hemagglutinin polypeptide is reconstituted intoanother set of liposomes containing another half of a reporter. When thetwo liposome populations are mixed together, fusion is detected byreconstitution of the reporter, for example, an enzymatic reaction thatcan be detected colorimetrically. An antibody inhibits fusion ifreporter activity is reduced or inhibited compared to reporter activityin an assay conducted in the absence of antibody or in the presence of acontrol antibody.

5.8.2 Antiviral Assays

An antibody or a composition thereof can be assessed in vitro forantiviral activity. In one embodiment, an antibody or compositionthereof is tested in vitro for its effect on growth of an Influenzavirus. Growth of Influenza virus can be assessed by any method known inthe art or described herein (e.g. in cell culture). In a specificembodiment, cells are infected at a MOI of 0.0005 and 0.001, 0.001 and0.01, 0.01 and 0.1, 0.1 and 1, or 1 and 10, or a MOI of 0.0005, 0.001,0.005, 0.01, 0.05, 0.1, 0.5, 1, 5 or 10 and incubated with serum freemedia supplemented a monoclonal antibody described herein or generatedin accordance with the methods provided herein Viral titers aredetermined in the supernatant by hemagglutinin plaques or any otherviral assay described herein. Cells in which viral titers can beassessed include, but are not limited to, MDCK cells, EFK-2 cells, Verocells, primary human umbilical vein endothelial cells (HUVEC), H292human epithelial cell line and HeLa cells. In vitro assays include thosethat measure altered viral replication (as determined, e.g., by plaqueformation) or the production of viral proteins (as determined, e.g., byWestern blot analysis) or viral RNAs (as determined, e.g., by RT-PCR ornorthern blot analysis) in cultured cells in vitro using methods whichare well known in the art or described herein.

In one non-limiting example, a monolayer of the target mammalian cellline is infected with different amounts (e.g., multiplicity of 3 plaqueforming units (pfu) or 5 pfu) of Influenza virus and subsequentlycultured at 37° C. in the presence or absence of various dilutions of amonoclonal antibody described herein or generated in accordance with themethods provided herein (e.g., 0.1 μg/ml, 1 μg/ml, 5 μg/ml, or 10μg/ml). Cultures are overlaid with agar and harvested 48 hours or 72hours post infection and titered by standard plaque assays known in theart on the appropriate target cell line (e.g., MDCK cells).

In a non-limiting example of a hemagglutination assay, cells arecontacted with an antibody and are concurrently or subsequently infectedwith the virus (e.g., at an MOI of 1) and the virus is incubated underconditions to permit virus replication (e.g., 20-24 hours). The antibodyis preferably present throughout the course of infection. Viralreplication and release of viral particles is then determined byhemagglutination assays using 0.5% chicken red blood cells. See, e.g.,Kashyap et al., PNAS USA 105: 5986-5991. In some embodiments, anantibody or composition thereof is considered an inhibitor of viralreplication if it reduces viral replication by at least 2 wells of HA,which equals approximately a 75% reduction in viral titer. In specificembodiments, an inhibitor reduces viral titer in this assay by 50% ormore, by 55% or more, by 60% or more, by 65% or more, by 70% or more, by75% or more, by 80% or more, by 85% or more, by 90% or more, or by 95%or more. In other specific embodiments, an inhibitor reduces viral titerin this assay by 1 log or more, approximately 2 logs or more,approximately 3 logs or more, approximately 4 logs or more,approximately 5 logs or more, approximately 6 logs or more,approximately 7 logs or more, approximately 8 logs or more,approximately 9 logs or more, approximately 10 logs or more, 1 to 5logs, 2 to 10 logs, 2 to 5 logs, or 2 to 10 logs.

5.8.3 Cytotoxicity Assays

Many assays well-known in the art can be used to assess viability ofcells (infected or uninfected) or cell lines following exposure to anantibody or composition thereof and, thus, determine the cytotoxicity ofthe antibody or composition thereof. For example, cell proliferation canbe assayed by measuring Bromodeoxyuridine (BrdU) incorporation (See,e.g., Hoshino et al., 1986, Int. J. Cancer 38, 369; Campana et al.,1988, J. Immunol. Meth. 107:79), (3H) thymidine incorporation (See,e.g., Chen, J., 1996, Oncogene 13:1395-403; Jeoung, J., 1995, J. Biol.Chem. 270:18367 73), by direct cell count, or by detecting changes intranscription, translation or activity of known genes such asproto-oncogenes (e.g., fos, myc) or cell cycle markers (Rb, cdc2, cyclinA, D1, D2, D3, E, etc). The levels of such protein and mRNA and activitycan be determined by any method well known in the art. For example,protein can be quantitated by known immunodiagnostic methods such asELISA, Western blotting or immunoprecipitation using antibodies,including commercially available antibodies. mRNA can be quantitatedusing methods that are well known and routine in the art, for example,using northern analysis, RNase protection, or polymerase chain reactionin connection with reverse transcription. Cell viability can be assessedby using trypan-blue staining or other cell death or viability markersknown in the art. In a specific embodiment, the level of cellular ATP ismeasured to determined cell viability.

In specific embodiments, cell viability is measured in three-day andseven-day periods using an assay standard in the art, such as theCellTiter-Glo Assay Kit (Promega) which measures levels of intracellularATP. A reduction in cellular ATP is indicative of a cytotoxic effect. Inanother specific embodiment, cell viability can be measured in theneutral red uptake assay. In other embodiments, visual observation formorphological changes may include enlargement, granularity, cells withragged edges, a filmy appearance, rounding, detachment from the surfaceof the well, or other changes. These changes may be given a designationof T (100% toxic), PVH (partially toxic—very heavy—80%), PH (partiallytoxic—heavy—60%), P (partially toxic—40%), Ps (partiallytoxic—slight—20%), or 0 (no toxicity—0%), conforming to the degree ofcytotoxicity seen. A 50% cell inhibitory (cytotoxic) concentration(IC₅₀) is determined by regression analysis of these data.

In a specific embodiment, the cells used in the cytotoxicity assay areanimal cells, including primary cells and cell lines. In someembodiments, the cells are human cells. In certain embodiments,cytotoxicity is assessed in one or more of the following cell lines:U937, a human monocyte cell line; primary peripheral blood mononuclearcells (PBMC); Huh7, a human hepatoblastoma cell line; 293T, a humanembryonic kidney cell line; and THP-1, monocytic cells. In certainembodiments, cytotoxicity is assessed in one or more of the followingcell lines: MDCK, MEF, Huh 7.5, Detroit, or human tracheobronchialepithelial (HTBE) cells.

An antibody or composition thereof can be tested for in vivo toxicity inanimal models. For example, animal models, described herein and/orothers known in the art, used to test the activities of an antibody orcomposition thereof can also be used to determine the in vivo toxicityof these antibodies. For example, animals are administered a range ofconcentrations of an antibody. Subsequently, the animals are monitoredover time for lethality, weight loss or failure to gain weight, and/orlevels of serum markers that may be indicative of tissue damage (e.g.,creatine phosphokinase level as an indicator of general tissue damage,level of glutamic oxalic acid transaminase or pyruvic acid transaminaseas indicators for possible liver damage). These in vivo assays may alsobe adapted to test the toxicity of various administration mode and/orregimen in addition to dosages.

The toxicity and/or efficacy of an antibody or composition thereof canbe determined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., for determining the LD₅₀ (the dose lethal to50% of the population) and the ED₅₀ (the dose therapeutically effectivein 50% of the population). The dose ratio between toxic and therapeuticeffects is the therapeutic index and it can be expressed as the ratioLD₅₀/ED₅₀. An antibody or composition thereof that exhibits largetherapeutic indices is preferred. While an antibody or compositionthereof that exhibits toxic side effects may be used, care should betaken to design a delivery system that targets such agents to the siteof affected tissue in order to minimize potential damage to uninfectedcells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage of an antibody or compositionthereof for use in humans. The dosage of such antibodies lies preferablywithin a range of circulating concentrations that include the ED₅₀ withlittle or no toxicity. The dosage may vary within this range dependingupon the dosage form employed and the route of administration utilized.For an antibody or composition thereof used in a method describedherein, the effective dose can be estimated initially from cell cultureassays. A dose may be formulated in animal models to achieve acirculating plasma concentration range that includes the IC₅₀ (i.e., theconcentration of the antibody that achieves a half-maximal inhibition ofsymptoms) as determined in cell culture. Such information can be used tomore accurately determine useful doses in humans. Levels in plasma maybe measured, for example, by high-performance liquid chromatography.Additional information concerning dosage determination is providedherein.

Further, any assays known to those skilled in the art can be used toevaluate the prophylactic and/or therapeutic utility of an antibody orcomposition thereof, for example, by measuring viral infection or acondition or symptoms associated therewith.

5.8.4 Assays for Measuring Antiviral Activity In Vivo

Antibodies and compositions thereof are preferably assayed in vivo forthe desired therapeutic or prophylactic activity prior to use in humans.For example, in vivo assays can be used to determine whether it ispreferable to administer an antibody or composition thereof and/oranother therapy. For example, to assess the use of an antibody orcomposition thereof to prevent an Influenza virus disease, the antibodyor composition can be administered before the animal is infected withInfluenza virus. Alternatively, or in addition, an antibody orcomposition thereof can be administered to the animal at the same timethat the animal is infected with Influenza virus. To assess the use ofan antibody or composition thereof to treat an Influenza virus infectionor disease associated therewith, the antibody or composition may beadministered after infecting the animal with Influenza virus. In aspecific embodiment, an antibody or composition thereof is administeredto the animal more than one time.

Antibodies and compositions thereof can be tested for antiviral activityin animal model systems including, but are not limited to, rats, mice,chicken, cows, monkeys, pigs, goats, sheep, dogs, rabbits, guinea pigs,etc. In a specific embodiment, an antibody or composition thereof istested in a mouse model system. Such model systems are widely used andwell-known to the skilled artisan. Non-limiting examples of animalmodels for Influenza virus are provided in this section.

In general, animals are infected with Influenza virus and concurrentlyor subsequently treated with an antibody or composition thereof, orplacebo. Alternatively, animals are treated with an antibody orcomposition thereof or placebo and subsequently infected with Influenzavirus. Samples obtained from these animals (e.g., serum, urine, sputum,semen, saliva, plasma, or tissue sample) can be tested for viralreplication via well known methods in the art, e.g., those that measurealtered viral titers (as determined, e.g., by plaque formation), theproduction of viral proteins (as determined, e.g., by Western blot,ELISA, or flow cytometry analysis) or the production of viral nucleicacids (as determined, e.g., by RT-PCR or northern blot analysis). Forquantitation of virus in tissue samples, tissue samples are homogenizedin phosphate-buffered saline (PBS), and dilutions of clarifiedhomogenates are adsorbed for a time period (e.g., 20 minutes or 1 hour)at 37° C. onto monolayers of cells (e.g., Vero, CEF or MDCK cells). Inother assays, histopathologic evaluations are performed after infection,preferably evaluations of the organ(s) the virus is known to target forinfection. Virus immunohistochemistry can be performed using aviral-specific monoclonal antibody.

The effect of an antibody or composition thereof on the infectiousdisease process or pathogenicity of a given virus can also be determinedusing in vivo assays in which the titer of the virus in an infectedsubject administered an antibody or composition thereof, the length ofsurvival of an infected subject administered an antibody or compositionthereof, the immune response in an infected subject administered anantibody or composition thereof, the number, duration and/or severity ofthe symptoms in an infected subject administered an antibody orcomposition thereof, and/or the time period before onset of one or moresymptoms in an infected subject administered an antibody or compositionthereof, is assessed. Techniques known to one of skill in the art can beused to measure such effects.

Influenza virus animal models, such as ferret, mouse, guinea pig, andchicken, developed for use to test antiviral agents against Influenzavirus have been described. See, e.g., Sidwell et al., Antiviral Res.,2000, 48:1-16; Lowen A. C. et al. PNAS., 2006, 103: 9988-92; andMcCauley et al., Antiviral Res., 1995, 27:179-186. For mouse models ofInfluenza, non-limiting examples of parameters that can be used to assayantiviral activity of antibodies administered to the Influenza-infectedmice include pneumonia-associated death, serum al-acid glycoproteinincrease, animal weight, lung virus assayed by hemagglutinin, lung virusassayed by plaque assays, and histopathological change in the lung.Statistical analysis is carried out to calculate significance (e.g., a Pvalue of 0.05 or less).

In yet other assays, histopathologic evaluations are performed afterinfection of an animal model subject. Nasal turbinates and trachea maybe examined for epithelial changes and subepithelial inflammation. Thelungs may be examined for bronchiolar epithelial changes andperibronchiolar inflammation in large, medium, and small or terminalbronchioles. The alveoli are also evaluated for inflammatory changes.The medium bronchioles are graded on a scale of 0 to 3+ as follows: 0(normal: lined by medium to tall columnar epithelial cells with ciliatedapical borders and basal pseudostratified nuclei; minimal inflammation);1+ (epithelial layer columnar and even in outline with only slightlyincreased proliferation; cilia still visible on many cells); 2+(prominent changes in the epithelial layer ranging from attenuation tomarked proliferation; cells disorganized and layer outline irregular atthe luminal border); 3+ (epithelial layer markedly disrupted anddisorganized with necrotic cells visible in the lumen; some bronchiolesattenuated and others in marked reactive proliferation).

The trachea is graded on a scale of 0 to 2.5+ as follows: 0 (normal:Lined by medium to tall columnar epithelial cells with ciliated apicalborder, nuclei basal and pseudostratified. Cytoplasm evident betweenapical border and nucleus. Occasional small focus with squamous cells);1+ (focal squamous metaplasia of the epithelial layer); 2+(diffusesquamous metaplasia of much of the epithelial layer, cilia may beevident focally); 2.5+ (diffuse squamous metaplasia with very few ciliaevident).

Virus immunohistochemistry is performed using a viral-specificmonoclonal antibody (e.g. NP-, N- or HN-specific monoclonal antibodies).Staining is graded 0 to 3+ as follows: 0 (no infected cells); 0.5+ (fewinfected cells); 1+ (few infected cells, as widely separated individualcells); 1.5+ (few infected cells, as widely separated singles and insmall clusters); 2+ (moderate numbers of infected cells, usuallyaffecting clusters of adjacent cells in portions of the epithelial layerlining bronchioles, or in small sublobular foci in alveoli); 3+(numerous infected cells, affecting most of the epithelial layer inbronchioles, or widespread in large sublobular foci in alveoli).

In one example, the ability to induce lung lesions and cause infectionin an animal model of virus infection is compared using wild-type virusand mock virus. Lung lesions can be assessed as a percentage of lunglobes that are healthy by visual inspection. Animals are euthanized 5days p.i. by intravenous administration of pentobarbital, and theirlungs are removed in toto. The percentage of the surface of eachpulmonary lobe that is affected by macroscopic lesions is estimatedvisually. The percentages are averaged to obtain a mean value for the 7pulmonary lobes of each animal. In other assays, nasal swabs can betested to determine virus burden or titer. Nasal swabs can be takenduring necropsy to determine viral burden post-infection.

In one embodiment, virus is quantified in tissue samples. For example,tissue samples are homogenized in phosphate-buffered saline (PBS), anddilutions of clarified homogenates adsorbed for 1 h at 37° C. ontomonolayers of cells (e.g., MDCK cells). Infected monolayers are thenoverlaid with a solution of minimal essential medium containing 0.1%bovine serum albumin (BSA), 0.01% DEAE-dextran, 0.1% NaHCO₃, and 1%agar. Plates are incubated 2 to 3 days until plaques could bevisualized. Tissue culture infectious dose (TCID) assays to titratevirus from PR8-infected samples are carried out as follows. Confluentmonolayers of cells (e.g., MDCK cells) in 96-well plates are incubatedwith log dilutions of clarified tissue homogenates in media. Two tothree days after inoculation, 0.05-ml aliquots from each well areassessed for viral growth by hemagglutination assay (HA assay).

In a specific embodiment, the ability of an antibody or compositionthereof to treat an Influenza virus infection or disease associatedtherewith is assessed by determining the ability of the antibody toconfer passive immunity to an Influenza virus disease in a subject. Theability of a monoclonal antibody described herein or generated inaccordance with the methods provided herein to confer passive immunityto an Influenza virus disease in a subject can be assessed using anymethods known in the art or described herein (see, e.g., Section 6.2infra).

5.8.5 Assays in Humans

In one embodiment, an antibody or composition thereof that modulatesreplication of an Influenza virus is assessed in infected humansubjects. In accordance with this embodiment, an antibody or compositionthereof is administered to the human subject, and the effect of theantibody and/or composition on viral replication is determined by, e.g.,analyzing the level of the virus or viral nucleic acids in a biologicalsample (e.g., serum or plasma). An antibody or composition thereof thatalters virus replication can be identified by comparing the level ofvirus replication in a subject or group of subjects treated with acontrol antibody to that in a subject or group of subjects treated withan antibody or composition thereof. Alternatively, alterations in viralreplication can be identified by comparing the level of the virusreplication in a subject or group of subjects before and after theadministration of an antibody or composition thereof. Techniques knownto those of skill in the art can be used to obtain the biological sampleand analyze the mRNA or protein expression.

In another embodiment, the effect of an antibody or composition thereofon the severity of one or more symptoms associated with an Influenzavirus infection/disease are assessed in an infected subject. Inaccordance with this embodiment, an antibody or composition thereof or acontrol antibody is administered to a human subject suffering fromInfluenza virus infection and the effect of the antibody or compositionon one or more symptoms of the virus infection is determined. Anantibody or composition thereof that reduces one or more symptoms can beidentified by comparing the subjects treated with a control antibody tothe subjects treated with the antibody or composition. Techniques knownto physicians familiar with infectious diseases can be used to determinewhether an antibody or composition thereof reduces one or more symptomsassociated with the Influenza virus disease.

5.9 Kits

Provided herein is a pharmaceutical pack or kit comprising one or morecontainers filled with one or more of the ingredients of thepharmaceutical compositions described herein, such as one or moreantibodies provided herein. Optionally associated with such container(s)can be a notice in the form prescribed by a governmental agencyregulating the manufacture, use or sale of pharmaceuticals or biologicalproducts, which notice reflects approval by the agency of manufacture,use or sale for human administration.

The kits encompassed herein can be used in the above methods. In oneembodiment, a kit comprises an antibody described herein, preferably anisolated antibody, in one or more containers. In a specific embodiment,the kits encompassed herein contain an isolated Influenza virus antigenthat the antibodies encompassed herein react with (e.g., the antibodybinds to the antigen) as a control. In a specific embodiment, the kitsprovided herein further comprise a control antibody which does not reactwith an Influenza virus antigen (e.g., the antibody does not bind to theantigen) that an antibody encompassed herein reacts with. In anotherspecific embodiment, the kits provided herein contain a means fordetecting the binding of an antibody to an Influenza virus antigen thatan antibody encompassed herein reacts with (e.g., the antibody may beconjugated to a detectable substrate such as a fluorescent compound, anenzymatic substrate, a radioactive compound, a luminescent compound, oranother antibody that is conjugated to a detectable substrate (e.g., theantibody may be conjugated to a second antibody which recognizes/bindsto the first antibody)). In specific embodiments, the kit may include arecombinantly produced or chemically synthesized Influenza virusantigen. In other specific embodiments, the kit may include as theInfluenza virus antigen the long alpha-helix of HA2 of an Influenzavirus (e.g., the hemagglutinin polypeptide of the Influenza virus strainA/Hong Kong/1/1968 (H3)). In certain specific embodiments, the kit mayinclude as the Influenza virus antigen amino acid residues within therange of 330 to 513, 345 to 513, 359 to 513, 360 to 513, 375 to 513, 390to 513, 384 to 439, 405 to 435, and/or 405 to 513 of the hemagglutininpolypeptide of the Influenza virus strain A/Hong Kong/1/1968 (H3) (i.e.,amino acids 1-184, 16-184, 30-184, 31-184, 46-184, 61-184, 70-110,76-106, and/or 76-184 of the hemagglutinin polypeptide numberedaccording to the classic H3 subtype numbering system). In a specificembodiment, a kit comprises amino acid residues 76-106 of thehemagglutinin polypeptide of the Influenza virus strain A/HongKong/1/1968 (H3) numbered according to the classic H3 subtype numberingsystem. In certain embodiments, a kit includes a virus vectorcomprising/generated to express amino acid residues 330 to 513, 345 to513, 359 to 513, 360 to 513, 375 to 513, 390 to 513, 384 to 439, 405 to435, and/or 405 to 513 of the hemagglutinin polypeptide of the Influenzavirus strain A/Hong Kong/1/1968 (H3) (i.e., amino acids 1-184, 16-184,30-184, 31-184, 46-184, 61-184, 70-110, 76-106, and/or 76-184 of thehemagglutinin polypeptide numbered according to the classic H3 subtypenumbering system) and/or amino acid residues 76-106 of the hemagglutininpolypeptide of the Influenza virus strain A/Hong Kong/1/1968 (H3)numbered according to the classic H3 subtype numbering system. TheInfluenza virus antigen provided in the kit may also be attached to asolid support. In a more specific embodiment the detecting means of theabove described kit includes a solid support to which an Influenza virusantigen is attached. Such a kit may also include a non-attachedreporter-labeled anti-human antibody. In this embodiment, binding of theantibody to the Influenza virus antigen can be detected by binding ofthe said reporter-labeled antibody.

6. EXAMPLES

The following examples are offered by way of illustration, and not byway of limitation.

6.1 Generation of Cross-Reactive Neutralizing Antibodies 6.1.1 PlasmidPreparation

Three DNA plasmids were generated encoding the hemagglutinin (HA)molecules from each of three antigenically distinct Influenza A virus H3subtypes: A/Hong Kong/1/1968 (plasmid 1), A/Alabama/1/1981 (plasmid 2),and A/Beijing/47/1992 (plasmid 3). To do so, the open reading frame ofHA from each virus was amplified from the parental virus strain andcloned into pCAGGS (Niwa et al., 1991, Gene 108: 1993-199), a mammalianexpression vector containing a chicken actin promoter that supportsprotein expression in mammalian cells.

Competent cells were transformed with either plasmid 1, plasmid 2, orplasmid 3 and positive transformants, i.e., cells harboring eitherplasmid 1, plasmid 2, or plasmid 3, were selected via resistance toampicillin. Positive transformants then were cultured and plasmids werepurified using the Qiagen Maxiprep Plasmid Purification Kit (Qiagen,Inc., Valencia, Calif.) according to the manufacturer's instructions.Plasmid DNA then was sequenced using methods known in the art to confirmsequence identity of the HA molecule in each of plasmids 1-3.

Once it was confirmed that the nucleotide sequence coding for HAmolecules in each of plasmids 1-3 was accurate, plasmid DNA was preparedat a concentration of 1.0 μg/μl in PBS (whole plasmid). Thisconcentration of plasmid DNA was used in subsequent immunizations ofmice.

6.1.2 Immunization

Ten C57/BL6 mice and ten Balb/c mice were immunized. Three days beforethe first immunization, mice were injected with 0.5% bupivacaine in thecalf muscle. Each immunization took place three weeks apart andconsisted of an intra-muscular (calf muscle) injection of 100 μl of eachplasmid preparation (plasmids 1-3) according to the followingimmunization schedule:

1. Immunization with plasmid 1.

2. Immunization with plasmid 2.

3. Immunization with plasmid 3.

Three weeks after the immunization with plasmid 3, the mice wereimmunized with Influenza virus strain A/Wyoming/3/2003, prepared asfollows: virus was grown in 10-day old chicken eggs and subsequentlyconcentrated and purified by centrifugation through a sucrose cushion.Virus then was resuspended in PBS. Protein concentration was determinedusing the Bradford assay and total protein was diluted to aconcentration of 250 μg/ml. Mice were injected with 200 μl of the virusin PBS, corresponding to 50 μg of inactivated virus. Two weeks aftereach immunization, serum from each mouse was evaluated to confirmeffective immunization.

6.1.3 Monoclonal Antibody Generation

Monoclonal antibodies were produced using hybridoma techniques known inthe art (see, e.g., Harlow et al., Antibodies: A Laboratory Manual,(Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling, et al.,in: Monoclonal Antibodies and T-Cell Hybridomas 563 681 (Elsevier, N.Y.,1981)). Briefly, spleens from the immunized mice were harvested andsplenocytes were isolated. The splenocytes then were fused to myelomacells. After fusion, hybridomas resulting from the fusion weredistributed in 96-well plates (10 plates per mouse, approximately 5viable hybridomas per well). Supernatants were harvested 1 weekpost-fusion (to allow for sufficient antibody production) and screenedby blot and by ELISA for reactivity with Influenza A virus strain A/HongKong/1/1968. Screening took place in iterative rounds alternating withsubcloning of positive wells until monoclonal cell populations wereobtained that had activity against A/Hong Kong/1/1968 as measured byeither blot assay or ELISA.

For the ELISA, wells of an ELISA plate were coated with 5 μg/ml ofpurified virus in PBS and the plate was incubated either for 3 hours at37° C. or overnight at 4° C. Prior to the assay, the purified virus wasremoved from the wells and 1% BSA in PBS was added for 30 minutes atroom temperature. The wells were subsequently washed three times with0.05% Tween 20 in PBS, followed by addition of the hybridoma supernatant(diluted 1:2) and incubation for 3 hours at 37° C. or overnight at 4° C.The wells were subsequently washed three times with 0.05% Tween 20 inPBS and anti-mouse IgG conjugated to alkaline phosphatase diluted in 1%BSA in PBS was added to each well followed by incubation for 3 hours at37° C. or overnight at 4° C. After incubation, the wells were washedthree times with 0.05% Tween 20 in PBS, followed by addition ofp-nitrophenylphospante substrate to each well. The reactions wereallowed to develop in the wells and reactivity of the hybridomasupernatants with Influenza A virus strain A/Hong Kong/1/1968 wasdetermined.

For assessment of reactivity by blot assay, purified virus was absorbedonto nitrocellulose membranes. The membranes then were blocked with 1%BSA in PBS for 30 minutes at room temperature followed by incubation ofthe membrane with the hybridoma supernatant (diluted 1:2) for 1 hour atroom temperature, with rocking. The membrane then was washed three timeswith 0.05% Tween 20 in PBS followed by incubation of the membrane withanti-mouse IgG conjugated to horseradish peroxidase diluted in 1% BSA inPBS for 1 hour at room temperature. The membrane then was washed threetimes with 0.05% Tween 20 in PBS followed by addition ofchemiluminescent substrate. The reactivity of the hybridoma supernatantswith Influenza A virus strain A/Hong Kong/1/1968 was then assessed byWestern blot using standard techniques (see, e.g., Current Protocols inProtein Science, Sean Gallagher, Hoefer Scientific Instruments, SanFrancisco, Calif., 1996).

Supernatants having activity in either assay against A/Hong Kong/1/1968were subcloned and monoclonal antibodies were isolated.

The ability of the monoclonal antibodies to bind various Influenza Avirus strains of the H3 subtype then was assessed by ELISA or Westernblot. As shown in FIG. 1A, purified monoclonal antibodies 7A7 and 39A4both bind to A/Hong Kong/1/1968 (H3) as assessed by ELISA. The purifiedmonoclonal antibody 12D1 binds to A/Hong Kong/1/1968 (H3) as assessed byWestern blot. In addition, the purified monoclonal antibody 12D1 bindsstrain A/Panama/2007/1999 (H3) (FIG. 1B) as assessed by Western blot;and monoclonal antibodies 7A7 and 39A4 bind strains A/Hong Kong/1/1968(H3), A/Panama/2007/1999 (H3), and A/Wisconsin/67/2005 (H3) (FIGS. 1Cand 1D) as assessed by ELISA.

6.1.4 Microneutralization Assay

The ability of the monoclonal antibodies generated in Section 6.1.3 toneutralize Influenza A viruses of the H3 subtype was assessed using amicroneutralization assay.

Viruses for use as neutralization targets were generated as follows:three stable cell lines were generated using MDCK cells, each expressingthe hemagglutinin of one of the following viruses: A/Hong Kong/1/1968,A/Panama/2007/1999, A/Wisconsin/67/2005. The cell lines were infectedwith a virus containing 7 segments (all but HA) from A/WSN/33 and onesegment coding for renilla luciferase (i.e., pseudotyped virus).Infection of HA-expressing cells with the WSN-luciferase virus yieldedvirus expressing the HA from the stable cell line and coding for renillaluciferase (to be used as a readout for infection in themicroneutralization assay).

The monoclonal antibodies then were evaluated for their ability toprevent infection of MDCK cells by the pseudotyped viruses; the readoutbeing luciferase activity, as detailed below.

In a 96-well plate, the monoclonal antibodies were serially diluted intwo-fold dilutions in PBS. The final volume of antibody/PBS in each wellwas 55 μl. Five μl of 50 pfu/ml of pseudotyped virus expressing eitherthe hemagglutinin from A/Hong Kong/1/1968, A/Panama/2007/1999 orA/Wisconsin/67/2005 diluted in PBS was added to each well, and thevirus/antibody mixture was incubated at 37° C. for 30 minutes. After the30 minutes, 50 μl of the virus/antibody mixture was removed and added toa 96-well plate seeded to confluency with MDCK cells. The cells werecultured with the virus/antibody mixture for 8-20 hours at 37° C.Following culture, the wells were rinsed and the cells were lysed.Luciferase activity then was measured by reading the relative lightunits using a luminometer. According to this assay, lack of luminescenceis indicative of no infection by the virus and therefore neutralizationof the virus by the monoclonal antibody.

As shown in FIG. 2, monoclonal antibodies 7A7, 39A4, and 12D1 all arecapable of neutralizing both the A/Hong Kong/1/1968 Influenza A virusstrain (FIG. 2A) and the A/Panama/2007/1999 Influenza A virus strain(FIG. 2B).

6.1.5 Plaque Reduction Assay

The ability of the monoclonal antibodies generated in Section 6.1.3 toneutralize Influenza A viruses of the H3 subtype was further assessedusing a plaque reduction assay.

Various dilutions of monoclonal antibody were incubated in a 96-wellplate with virus (˜50 pfu/well) for 1 hour, with rocking. Thevirus/antibody mixture (200 μl) was subsequently added to MDCK cellsgrown to confluency. The cells and virus/antibody mixture were incubatedfor 20 minutes at 37° C. and subsequently the virus/antibody mixture wasaspirated from the cells and the cells were overlaid with agar having aconcentration of antibody identical to the concentration of antibody inthe virus/antibody mixture being analyzed. The plates then wereincubated for 3 days at 37° C. to allow for plaque formation.

As shown in FIG. 3, monoclonal antibodies 7A7, 12D1, and 39A4 neutralizeall Influenza A virus strains of the H3 subtype tested, i.e., A/HongKong/1/1968 (H3), A/Beijing/47/1992 (H3), A/Pan/2007/1999 (H3), andBRIS/07 (H3) but do not neutralize Influenza A virus strains of subtypesother than H3 that were tested, i.e., New CAL/99 (H1), DK/64 (H4), andTKY/63 (H7).

6.1.6 Red Blood Cell Fusion Assay

The ability of the monoclonal antibodies generated in Section 6.1.3 toinhibit fusion between Influenza A viruses of the H3 subtype and hostcells was assessed using a red blood cell fusion assay.

Virus was incubated with chicken red blood cells (0.2% finalconcentration RBCs) for ten minutes on ice followed by addition ofmonoclonal antibody. The mixture of virus, cells, and antibody wasincubated on ice for 30 minutes followed by addition of sodium citratebuffer, pH 4.4, and incubation at room temperature for 5-30 minutes. 200μl of the virus, cells, and antibody mixture was removed at various timepoints, centrifuged for 3 minutes at 3000 rpm, and the supernatant wastransferred to a 96-well ELISA plate and read at OD₄₁₀. Positivereadings indicated the presence of heme in the supernatant, whichindicates that lysis of red blood cells occurred due to a low-pH fusionreaction between the cells and virus, particularly between thehemagglutinin protein of the virus and the cell.

As shown in FIG. 4, monoclonal antibodies 7A7 and 12D1 inhibit low-pHfusion of A/Hong Kong/1/1968 (H3) hemagglutinin and red blood cells.

6.1.7 Conclusions

The method of generating cross-reactive neutralizing monoclonalantibodies provided herein successfully results in the generation ofmonoclonal antibodies that cross-react with hemagglutinin from InfluenzaA virus strains of the H3 subtype that are antigenically distinct fromone another. Moreover, these antibodies are capable of neutralizing allof the Influenza A virus strains of the H3 subtype tested as determinedby multiple assays.

6.2 In Vivo Protection by Passive Transfer of Cross-ReactiveNeutralizing Antibodies

The ability of the cross-reactive neutralizing monoclonal antibodiesgenerated in accordance with the methods described herein to protectmice from challenge with Influenza A virus was assessed by passivetransfer of the antibodies in mice.

The ability of the cross-reactive neutralizing monoclonal antibodiesgenerated in accordance with the methods described herein to conferpassive immunity can be determined by assessing the ability of theantibodies to prolong the survival of mice infected with an Influenzavirus. According to this method, mice are administered a cross-reactiveneutralizing monoclonal antibody prior to challenge with an Influenzavirus, e.g., 1 hour prior to challenge. The duration of survival of themice is then calculated and compared to the survival of similarlychallenged mice that were administered a control, e.g., PBS, rather thana cross-reactive neutralizing monoclonal antibody.

As demonstrated in FIG. 5, male BALB/c mice, 5 mice per group, 6-8 weeksof age administered approximately 15 mg/kg monoclonal antibody 12D1 byintraperitoneal injection 1 hour prior to intranasal challenge with 10⁵pfu/ml X31 virus survive the viral challenge, whereas mice administeredPBS alone die seven days subsequent to viral challenge.

The ability of the cross-reactive neutralizing monoclonal antibodiesgenerated in accordance with the methods described herein to conferpassive immunity can be determined by assessing the ability of theantibodies to inhibit weight loss of mice infected with an Influenzavirus. According to this method, mice are administered a cross-reactiveneutralizing monoclonal antibody prior to challenge with an Influenzavirus, e.g., 1 hour prior to challenge. The weight loss of the mice isthen measured over time and compared to the weight loss of similarlychallenged mice that were administered a control, e.g., PBS, rather thana cross-reactive neutralizing monoclonal antibody.

As demonstrated in FIG. 7, passive transfer of the cross-reactiveneutralizing monoclonal antibodies 12D1 and 39A4 results in decreasedweight loss in mice challenged with Influenza A virus strain A/HongKong/1/1968 (H3) as compared to mice administered PBS, rather thanantibody, thus demonstrating generation of passive immunity in thesemice against the A/Hong Kong/1/1968 (H3) strain. Different groups ofmice (five mice per group) were administered monoclonal antibody 12D1(30 mg/kg), monoclonal antibody 39A4 (15 mg/kg or 30 mg/kg), or PBS byintraperitoneal injection 1 hour prior to intranasal challenge with 10⁵pfu/ml X31, a chimeric virus containing the hemagglutinin andneuramidase gene segments from A/Hong Kong/1/1968 (H3) and the six otherInfluenza virus genes segments (not hemagglutinin and neuramidase) fromthe murine Influenza A virus A/PR/8/34. The weights of the mice weremeasured each day and the average weight of the five mice in each groupis plotted in FIG. 7.

6.2.1 Conclusions

The cross-reactive neutralizing monoclonal antibodies generated inaccordance with the methods described herein are capable of conferringpassive immunity in vivo.

6.3 Determination of the Binding Region the Cross-Reactive NeutralizingAntibodies

To determine the binding region of the cross-reactive neutralizingmonoclonal antibodies generated in accordance with the methods describedherein, nucleic acid constructs were generated that encode a fusionprotein comprising the coding sequence of the green fluorescent protein(GFP) fused to different fragments of the A/Hong Kong/1/1968 (H3)hemagglutinin. HA fragments were generated by polymerase chain reactionand were subsequently cloned into pCAGGS-GFP vector. See FIG. 8 for adiagram of the fusion proteins encoded by the various nucleic acidconstructs. Each nucleic acid construct (50 ng/6-well dish) wastransfected into 293T cells using Lipofectamine 2000 (Invitrogen) andthe ability of a monoclonal antibody to bind to a fusion proteinexpressed by the cells was assessed by Western blot using standardtechniques (see, e.g., Current Protocols in Protein Science, SeanGallagher, Hoefer Scientific Instruments, San Francisco, Calif., 1996).Anti-GFP antibodies were used as a positive control for proteinexpression.

As shown in FIG. 9 the monoclonal antibody 12D1 binds to the HA2 regionof the hemagglutinin protein of Influenza A virus strain A/HongKong/1/1968 (H3) as demonstrated by the antibody's ability to bind tothe region of the A/Hong Kong/1/1968 (H3) hemagglutinin corresponding toamino acid residues 304 to 513 and small fragments thereof (FIG. 9B,lanes 5-10). In particular, the monoclonal antibody 12D1 is capable ofbinding to the region of A/Hong Kong/1/1968 (H3) hemagglutinincorresponding to amino acids 405 to 513 (FIG. 9B, lanes 10). Asexpected, the anti-GFP antibody bound to each construct having the GFPfusion (FIG. 9A, lanes 3-10), the anti-GFP antibody did not bind to theA/Hong Kong/1/1968 (H3) hemagglutinin (FIG. 9A, lane 2), and neithermonoclonal antibody 12D1 or the anti-GFP antibody bound to cell lysatesfrom untransfected cells (FIGS. 9A and 9B, lane 1).

6.4 Broadly Protective Anti-Influenza Antibodies 6.4.1 Materials andMethods 6.4.1.1 Animals

Six week old female BALB/c mice from Jackson Laboratory were used forall experiments.

6.4.1.2 Viruses and Cells

Madin Darby canine kidney cells from ATCC were used for all cell basedassays. Cells were maintained in minimum essential medium supplementedwith 10% fetal bovine serum, and 100 units/ml of penicillin-100 μg/ml ofstreptomycin. All viruses were propagated in eggs. Viruses used invarious studies: A/Hong Kong/1/1968 (HK/68) (H3), A/Alabama/1/1981(AL/81) (H3), A/Georgia/1981 (H3), A/Beijing/47/1992 (BJ/92) (H3),A/Wyoming/3/2003 (H3), A/Wisconsin/67/2005 (WI/05) (H3),A/Brisbane/102007 (BR/07) (H3), A/New York/2008 (NY08) (H3),A/Texas/36/1991 (TX/91) (H1), A/New Caledonia/20/99 (N.Cal/99) (H1),A/Duck/England/1962 (Dk/62) (H4), A/Turkey/England/1963 (Tky/63) (H7),A/Equine/Kentucky/2002 (e/KY/02) (H3), A/Ann Arbor/6/1960 (AA/60) (H2),A/Fort Monmouth/1/1947 (FM/47) (H1). Purified virus was prepared by highspeed centrifugation (43,000 rpm, 1 hour) of allantoic fluid through a20% sucrose cushion.

6.4.1.3 Antibody Preparations

Hybridoma supernatants were used for screening of mAbs for reactivity byenzyme-linked immunosorbent assay (ELISA) and by western blot. For otherassays, purified monoclonal antibody or ascites preparations treatedwith receptor-destroying enzyme (see, e.g., Jordan et al., J Immunol,1954; 72(3):229-35) were used. RDE—treated ascites was used formeasurement of binding by ELISA, microneutralization, plaque reductionand fusion assays. Antibodies were purified by methods previouslydescribed (see, e.g., Harlow E, Lane D. Antibodies: a laboratory manual.Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory; 1988. xiii, 726p.). Because of differences in isotypes, Protein A-agarose (Roche) wasused for purification of mAbs 7A7 and 39A4 while protein G-agarose(Roche) was used for purification of mAb 12D1.

6.4.1.4 Immunization of Mice and Hybridoma Production

Six-week old BALB/c mice were immunized with DNA constructs coding forthe open-reading frame of Influenza virus hemagglutinin in the pCAGGSplasmid (see, e.g., Basler et al., Proc Natl Acad Sci USA 98:2746-2751). Individual immunizations were given intramuscularly, 3-weeksapart and consisted of 100 ug DNA in 100 ul PBS. Hemagglutinins utilizedin the immunization schedule were cloned from the following parentalviruses—primary immunization: A/Hong Kong/1/1968, secondaryimmunization: A/Alabama/1/1981, tertiary immunization: A/Beijing/47/1992HA. Three days prior to fusion, mice were boosted with 50 ug purifiedA/Wyoming/3/2003 virus. B cell hybridomas were produced by methodspreviously described (see, e.g., de StGroth et al., J Immunol Methods35: 1-21).

6.4.1.5 Screening of Hybridoma Supernatants

Hybridoma supernatants were screened by blot and by ELISA for reactivitywith A/Hong Kong/1/1968 virus. For the ELISA, direct binding to wellscoated with 5 ug/ml purified virus, 50 ul per well was assessed. For theblot assay, 10 μg purified virus was adsorbed onto nitrocellulose stripsand individual strips were incubated with hybridoma supernatants. Forthe ELISA and blot assays, binding of antibody to virus was detectedusing goat anti-mouse γ-chain horse radish peroxidase secondary antibody(SouthernBiotech, Birmingham, Ala.). All wells that had activity ineither assay against A/Hong Kong/1/1968 virus were subcloned repeatedlyto ensure the monoclonality of the hybridoma populations.

6.4.1.6 Western Blots

Blots were produced by methods previously described (see, e.g., Towbinet al., Proc Natl Acad Sci USA 1979; 76(9):4350-4). Samples were boiledfor 5 minutes at 100° C. in loading buffer containing SDS and 0.6M DTT.SDS migration buffer was used for electrophoresis. For non-reducing gelconditions samples were prepared in loading buffer with SDS but withoutreducing agent and were not boiled.

6.4.1.7 Immunofluorescence Test

MDCK cells were infected with virus at a multiplicity of infection of 1and incubated for 6 hours at 37° C. Infected and uninfected cells wereincubated with 1 μg/ml mAb for 1 hour at room temperature. Goatanti-mouse fluorescein conjugate (SouthernBiotech) was used fordetection of mAb binding.

6.4.1.8 Microneutralization Assay

Two stable cell lines were generated that expressed the HA of A/HongKong/1/1968 virus or A/Panama/2007/1999 virus. Pseudotyped virusesexpressing the HA of either cell line were generated by infection ofcells with a virus that carries seven segments from A/WSN/33 virus (allexcept the HA segment) and one segment encoding Renilla luciferase.Pseudotyped viruses expressing the HA of A/Hong Kong/1/1968 virus orA/Panama/2007/1999 virus were used as the neutralization target. Viruseswere incubated with mAb at room temperature for 30 minutes, rockingPurified polyclonal mouse IgG (Invitrogen) was used for the negativecontrol. The mixture containing virus and mAb was then transferred towells of a 96-well plate seeded to confluency with MDCK cells andincubated for 12 hours at 37° C. Individual determinants were performedin triplicate. After incubation, luciferase activity in cell-lysates wasmeasured as a read-out of virus infection (Renilla luciferase assaysystem, Promega).

6.4.1.9 Plaque Reduction Assay

Antibody and virus (˜50 pfu/well) were co-incubated at room temperaturefor 30 minutes, rocking 6 well plates seeded with MDCK cells were washedonce with PBS and 200 μl of virus and mAb was added to each well thenincubated for 20 minutes, 37° C. Virus with mAb was aspirated from cellsand an agar overlay containing antibody was added to each well. Plateswere incubated for 3 days, 37° C. and plaques were counted by crystalviolet staining Purified mouse IgG (Invitrogen) was used for thenegative control.

6.4.1.10 Passive Transfer Experiments

Before infection, mice were anesthetized by intraperitonealadministration of a ketamine (75 mg/kg of body weight)/xylazine (15mg/kg of body weight) mixture. 6 week old BALB/c mice were given 30mg/kg mAb intraperitoneally either one hour before, 24 hours after or 48hours after challenge with 10 LD₅₀ A/Hong Kong/1/1968, A/PR/8/34reassortant virus or 2700 pfu A/Georgia/1981 virus (lung titerexperiment). Purified mouse IgG (Invitrogen) was used for the negativecontrol. Virus was suspended in PBS and administered intranasally in 50μl (25 μl per nostril). Mice were weighed daily and sacrificed if theyfell to 75% of starting weight. For the lung titer experiment, mouselungs were harvested 4 days post infection with A/Georgia/1981 and virustiters in lung homogenates were determined by plaque assay. Forhistologic evaluation of lung damage, lungs were harvested 4 days postinfection with A/Hong Kong/1/1968-A/PR/8/34 reassortant virus. Tissueswere imbedded in paraffin and sections were stained with hematoxylin andeosin.

6.4.1.11 Hemaglutinin Inhibition Assay and Fusion Assay

MAbs were tested in a standard hemagglutination inhibition assay (see,e.g., Cohen et al., Virology 1963; 20:518-29) using chicken red bloodcells and A/Hong Kong/1/1968 virus. For the red blood cell fusion assay,virus was incubated with chicken red blood cells (2% final red cellconcentration) on ice for 10 minutes. Dilutions of antibody were addedand samples were incubated on ice for 30 minutes. Sodium citrate buffer,pH 4.6 was then added to bring the final pH to 5.0 and samples wereincubated for 30 minutes at room temperature. Samples were centrifugedfor 3 minutes at 3000 rpm to pellet red blood cells and supernatantswere then transferred to an ELISA plate for determination of NADPHcontent by optical density measurement (340 nm). NADPH was present inthe supernatant as a function of fusion-induced red blood cell lysis.

6.4.1.12 Hemaglutinin Truncation Mutants

DNA constructs were generated in the pCAGGS plasmid that coded fortruncations of the A/HK/1/68 virus hemagglutinin fused to greenfluorescent protein. All constructs were sequenced and confirmed. 293Tcells were then transfected using Lipofectamine 2000 (Invitrogen, Inc.)with the various pCAGGS encoding the HA-GFP fusion gene. Cell lysateswere resolved in a 4-20% Tris-HCl SDS-PAGE gel (Bio-Rad Laboratories)and proteins were blotted onto a Protran nitrocellulose membrane(Whatman). GFP and truncated HA fragments were detected using rabbitanti-GFP (Santa Cruz Biotechnology, Inc.) and anti-H3 mAb 12D1respectively. Secondary antibodies were anti-rabbit IgG HRP (Dako) andanti-mouse Ig HRP(GE Healthcare).

6.4.2 Results 6.4.2.1 Isolation of Broadly-Reactive Anti-H3 mAbs

In order to enhance the production of cross-reactive antibodyspecificities, mice were immunized by sequential administration with DNAcoding for the hemagglutinin from H3 viruses arising approximately 10years apart: A/Hong Kong/1/1968, A/Alabama/1/1981, A/Beijing/47/1992.Three days prior to fusion, mice were boosted with the H3 virusA/Wyoming/3/2003. By performing the fusion rapidly after virus boost itwas ensured that only hemagglutinin-specific B cells were present in thespleen at time of fusion. The hemagglutinins chosen were from virusesthat arose over several decades, thus representing multiple H3 antigenicclusters (see, e.g., Smith et al., Science 2004; 305(5682):371-6).Post-fusion, hybridoma supernatants were screened for the ability tobind A/Hong Kong/1/1968 by Western blot or by ELISA and successiverounds of subcloning were performed on positive supernatants untilmonoclonal hybridoma populations were isolated.

The immunization schedule utilized successfully elicited the productionof antibodies with broad reactivity against H3 viruses. Approximately120 clones were isolated that reacted with A/Hong Kong/1/1968; of those,eight mAbs were cross-reactive against all of the H3 hemagglutininstested. The particular immunization protocol also preferentiallyelicited the production of antibodies specific for the HA2 subunit ofthe hemagglutinin. Of the 8 mAbs identified, 5 mAbs react with HA2 and 1mAb reacts with HA1 by Western blot. The remaining 2 mAbs (7A7 and 39A4)bind conformational epitopes present in the HA trimer as detected bywestern blot of purified H3 virus proteins separated under non-reducinggel conditions. All mAbs were reactive in a purified H3 virus ELISA.Three of the mAbs, 7A7, 12D1, 39A4, had the highest activity by ELISAand were selected for thorough characterization (Table 1, FIG. 10).

TABLE 1 Pattern of reactivity of anti-H3 mAbs. All mAbs have activity byELISA and all mAbs react by western blot under reducing conditionsexcept mAbs 7A7 and 39A4 that react with the HA trimer undernon-reducing conditions. All mAbs are negative for hemagglutinationinhibition activity at 50 ug/ml. Isotype ELISA WB HI 7A7 IgG2b + Trimer− 12D1 IgG1 + HA2 − 39A4 IgG2a + Trimer − 62F11 IgG2a + HA2 − 36A7IgG2b + HA2 − 66A6 IgG1 + HA1 − 49E12 IgG2b + HA2 − 21D12 IgG1 + HA2 −

Antibodies 7A7, 12D1 and 39A4 react by ELISA with purifiedA/Alabama/1/1981 and purified A/Hong Kong/1/1968 viruses (FIG. 11). MAbXY102 is specific for the hemagglutinin of A/Hong Kong/1/1968 virus.7A7, 12D1 and 39A4 show broad reactivity by immunofluorescence againstcells infected with all H3 viruses spanning 40 drift years. MAbs 7A7 and39A4 also react by immunofluorescence with other Influenza A viruseschosen at random, including representative H1, H2 and equine H3 viruses(Table 2).

TABLE 2 Reactivity of mAbs at 5 ug/ml by immunofluorescence against MDCKcells infected with a panel of randomly chosen viruses. MAb XY102 wasgenerated by immunization with A/HK/1968 (H3) virus and mAb 10C4 wasgenerated by immunization with A/TX/1991 (H1) virus. Virus Subtype 7A712D1 39A4 10C4 XY102 HK/68 H3 + + + − + AL/81 H3 + + + − − BJ/92H3 + + + − − WI/05 H3 + + + − − BR/07 H3 + + + − − NY/08 H3 + + + − −TX/91 H1 + − + + − FM/47 H1 + − + − − AA/60 H2 + − + − − Equine/ H3 +− + − − KY/02

6.4.2.2 mAbs Neutralize H3 Viruses Spanning 40 Drift Years

The anti-H3 mAbs were first evaluated for their ability to neutralize H3Influenza viruses by microneutralization assay. Viruses used in thisassay contain a gene segment coding for firefly luciferase in place ofthe viral hemagglutinin; a hemagglutinin is present on the viralenvelope due to propagation of virus in cells stably expressing aparticular H3 hemagglutinin protein. Luciferase viruses were generatedthat express the hemagglutinin of A/HK/1968 or A/Panama/99 viruses.Neutralization of viruses by anti-H3 mAbs was determined based onluciferase activity after single-cycle replication. Bymicroneutralization, the three anti-H3 mAbs were determined toneutralize the hemagglutinin of both A/HK/1968 and A/Pan/99 (FIG. 12).

Next, neutralization activity by plaque reduction assay was evaluated.The anti-H3 mAbs were able to prevent infection (not simply reduceplaque size) of Madin Darby canine kidney cells by H3 viruses arisingover 40 drift years: A/HK/1968, A/BJ/1992, A/Pan/99, A/Bris/07, A/NY/08(FIG. 13). Monoclonal antibodies 7A7, 12D1 and 39A4 were tested againstrepresentative H4 and H7 viruses (Group 2) as well as an H1 virus(Group 1) and it was determined that they did not neutralize thesenon-H3 subtype viruses (FIG. 13).

6.4.2.3 Anti-H3 mAbs in the Treatment of Influenza in Mice

The three mAbs were tested in vivo for use as passive transfer therapiesin disease caused by H3 virus infection. Mice were given 30 mg/kg mAbintraperitoneally either 1 hour before, 24 hours post or 48 hours postchallenge with 10 mouse LD₅₀ reassortant H3 virus (The A/HK/68reassortant virus contains the six non-hemagglutinin, non-neuraminidasesegments from the mouse-adapted A/PR/8 virus). Mice were weighed dailyand were sacrificed if they reached 75% of their starting weight.Treatment of mice with mAb 12D1 either prophylactically ortherapeutically was 100% protective. mAb 39A4 was evaluated for efficacyby prophylactic treatment and was similarly 100% protective in vivo.Mice treated prophylactically with mAb 7A7 were only 40% protectedagainst the A/HK/68 reassortant virus (FIG. 14).

Next, the effect of prophylactic treatment with mAb 12D1 or 39A4 on lungdamage caused by H3 viral pneumonia was assessed by histologicevaluation of tissue taken 4 days post infection with the A/HK/68reassortant virus. Without treatment, lungs showed degenerative changeswith focal hemorrhaging, dense neutrophilic infiltrates and diffusealveolar damage with edema. Treatment with either anti-H3 mAbsignificantly diminished pathologic changes (FIG. 15).

Having demonstrated protective activity in vivo against the A/HK/68reassortant virus, cross-protection mediated by mAbs 12D1 and 39A4against a second H3 virus, A/Georgia/1981, was evaluated. MAbs 12D1 and39A4 were administered as described above to BALB/c mice one hour priorto infection. Mice were then infected intranasally with 2700 pfuA/Georgia/1981 and lung titers were evaluated two days post infection.The anti-H3 mAbs reduced lung titers by 97.75% (12D1) or 99.03% (39A4)(FIG. 16).

6.4.2.4 Anti-H3 mAbs Act by Inhibiting Viral Fusion

In order to determine the mechanism of virus neutralization by theanti-H3 mAbs, the ability of the mAbs to inhibit virus hemagglutinationof chicken red blood cells was examined. None of the three mAbs hadhemagglutination inhibition activity, suggesting that the mAbs did notact by obstructing the binding of virus to the host-cell.

Next, the effect of the anti-H3 mabs on virus fusion was tested. MAbs7A7, 12D1 and 39A4 were determined to inhibit the low-pH fusion ofA/HK/1968 virus with chicken red blood cells by at least 80% at 10 ug/ml(FIG. 17).

6.4.2.5 Binding Epitope of mAb 12D1

The identity of the region of the H3 hemagglutinin that might elicitantibodies with fine specificities mirroring those of 12D1 or 39A4 wasexamined. Sixteen passages of A/HK/1968 virus in the presence of theanti-H3 mAbs 12D1 or 39A4 did not yield escape variants which might haveassisted in identification of the binding epitopes. The hemagglutinin ofsix plaques present after incubation of A/HK/1968 virus with 50 ug/mlmAb 12D1 or 39A4 in a plaque assay was sequenced and no changes from thewild-type hemagglutinin were found. Because mAb 12D1 mediates protectionagainst Influenza disease in vivo and reacts with a continuous epitopeof the viral hemagglutinin (no trimeric structure required), asevidenced by reactivity with the denatured hemagglutinin monomer byWestern blot (FIG. 10), the 12D1 binding epitope was focused on.Hemagglutinin truncation mutants consisting of hemagglutinin segments ofvarying length fused to GFP were generated. GFP expression was utilizedto assess expression of the constructs in transfected 293T cells. Byanalysis of the truncation mutants, it was determined that the 12D1paratope makes dominant interactions with the HA2 subunit in the regionof amino acids 30-106. Diminished 12D1 binding without diminished GFPexpression in the 76-184 and 91-184 truncations along with loss ofbinding with the 106-184 truncation suggested that 12D1 binding isdependent on contacts with amino acids in the HA2 76-106 region (FIG.18). These 30 amino acids fall within the membrane distal half of thelong alpha-helix of HA2. The 12D1 paratope may have additional contactswith amino acids outside of this region (in HA1 or HA2) that are notrequired for binding by Western blot.

6.4.3 Conclusion

An immunization schedule was developed that elicitedbroadly-neutralizing antibodies against H3 Influenza viruses in vitroand in vivo.

6.5 Cross-Reactive Anti-H3 Monoclonal Antibody 66A6

Monoclonal antibody 66A6 was generated using the same approach used forthe generation of antibodies 7A7, 12D1, and 39A4, as described inSection 6.1.

6.5.1 Antibody 66A6 Binds H3 Viruses

Using the ELISA approach described in Section 6.1.3., the ability ofmonoclonal antibody 66A6 to bind Influenza A virus strains A/HongKong/1/1968, A/Brisbane/10/2007, and A/Panama/2007/1999 was assessed. Asshown in FIG. 23, monoclonal antibody 66A6 bound each of Influenza virusstrains.

6.5.2 Hemaglutination-Inhibition Activity of Antibody 66A6

The ability of monoclonal antibody 66A6 to inhibit fusion of Influenza Avirus strain A/Hong Kong/1/1968 was assessed as described in Section6.1.6. As shown in FIG. 24, monoclonal antibody 66A6 does not inhibitthe low-pH fusion of A/Hong Kong/1/1968 hemagglutinin and red bloodcells.

6.5.3 Antibody 66A6 Binds the HA1 Portion of H3 Virus Hemaglutinin

The binding region of monoclonal antibody 66A6 was determined using theapproach described in Section 6.3. As shown in FIG. 25, monoclonalantibody 66A6 binds to the HA1 region of the hemagglutinin protein ofInfluenza virus strain A/Hong Kong/1/1968 (H3) as assessed by Westernblot.

6.5.4 Antibody 66A6 Neutralizes H3 Virus

A plaque reduction assay was used as described in Section 6.1.5 toassess the ability of monoclonal antibody 66A6 to neutralize Influenza Aviruses of the H3 subtype. As shown in FIG. 26, monoclonal antibody 66A6neutralizes Influenza A virus strains A/Panama/2007/1999 (H3) andA/Alabama/1/1981 (H3).

6.5.5 Passive Transfer of Antibody 66A6

The ability of monoclonal antibody 66A6 to protect mice from challengewith Influenza A virus was assessed. According to this method, BALB/cmice, in groups of five, were administered 20 mg of 66A6 monoclonalantibody (intraperitoneally—Group 1) or a control (PBS—Group 2) 1 hourprior to challenge with a 10LD50 X31 chimeric virus containing thehemagglutinin and neuramidase gene segments from A/Hong Kong/1/1968 (H3)and the six other Influenza virus genes segments (not hemagglutinin andneuramidase) from the murine Influenza A virus A/PR/8/34. The bodyweight of the mice was measured daily and the body weight of the miceadministered the 66A6 monoclonal antibody was compared to the bodyweight of the mice that were administered a control (PBS).

As demonstrated in FIG. 27, passive transfer of the cross-reactiveneutralizing monoclonal antibody 66A6 results in decreased weight lossin mice challenged with Influenza A virus strain X31 as compared to miceadministered PBS alone. The result demonstrates the generation ofpassive immunity in these mice against the X31 strain. The averageweight of the mice in each group is plotted in FIG. 27. Mice that fellto below 75% of their starting weight were sacrificed.

6.6 Cross-Reactive Anti-H1/H3 Monoclonal Antibodies 6.6.1 Immunizationand Hybridoma Generation

A cyclical immunization strategy was used to generate monoclonalantibodies that are cross-reactive to antigenically distinct H1 and H3subtypes of Influenza A virus (FIG. 30). Six-week old BALB/c mice wereimmunized with DNA constructs coding for the open-reading frame ofInfluenza virus hemagglutinin in the pCAGGS plasmid (see, e.g., Basleret al., Proc Natl Acad Sci USA 98: 2746-2751). Individual immunizationswere given intramuscularly, 3-weeks apart and consisted of 100 μg DNA in100 μl PBS. Hemagglutinins utilized in the immunization schedule werecloned from the following parental viruses—primary immunization: A/HongKong/1/1968 (H3), secondary immunization: A/USSR/92/77 (H1), tertiaryimmunization: A/California/1/88 (H3), quaternary immunization:A/California/04/09 (H1). Three weeks after the final immunization andthree days prior to generation of hybridomas, mice were boostedintravenously with a composition comprising 50 μg purifiedA/Brisbane/59/07-like (H1) virus and 50 μg purifiedA/Brisbane/10/07-like (H3) virus. B cell hybridomas were produced bymethods previously described (see, e.g., de StGroth et al., J ImmunolMethods 35: 1-21).

6.6.2 Screening of Hybridoma Supernatants

Hybridoma supernatants were screened for reactivity with A/HongKong/1/1968 virus and A/California/04/09 (H1) by ELISA as described inSection 6.4.1.5. Hybridomas that reacted with either strain wereselected for, including those that produce Antibody 1, Antibody 2,Antibody 3, and Antibody 4.

6.6.3 Immunofluorescence Test

MDCK cells were infected with virus at a multiplicity of infection of 1or 0.5, incubated for 12 hours at 37° C., and fixed in the absence oftrypsin. Infected and uninfected cells were incubated with 1 μg/ml mAbfor 1 hour at room temperature. Goat anti-mouse fluorescein conjugate(SouthernBiotech) was used for detection of mAb binding. Antibody 1 andAntibody 2 recognize HA from 3 H1 Influenza viruses byimmunofluorescence (FIG. 31). Antibody 4 recognizes the HA from two H3Influenza viruses by immunofluorescence (FIG. 31).

6.6.4 Western Blots

Western blots were produced by methods previously described (see, e.g.,Towbin et al., Proc Natl Acad Sci USA 1979; 76(9):4350-4). Samples wereboiled for 5 minutes at 100° C. in loading buffer containing SDS and0.6M DTT. SDS migration buffer was used for electrophoresis. Underdenaturing and reducing conditions, Antibody 1 and Antibody 3 do notrecognize HA (FIG. 32). Antibody 4 recognizes the HA2 subunit of HA(FIG. 32).

6.7 Cross-Reactive Anti-H1 Monoclonal Antibodies 6.7.1 Immunization andHybridoma Generation

A cyclical immunization strategy was used to generate monoclonalantibodies that are cross-reactive to antigenically distinct Influenza Avirus H1 subtypes (FIG. 33). Six-week old BALB/c mice were immunizedwith DNA constructs coding for the open-reading frame of Influenza virushemagglutinin in the pCAGGS plasmid (see, e.g., Basler et al., Proc NatlAcad Sci USA 98: 2746-2751). Individual immunizations were givenintramuscularly, 3-weeks apart and consisted of 100 μg DNA in 100 μlPBS. Hemagglutinins utilized in the immunization schedule were clonedfrom the following parental viruses—primary immunization: A/SouthCarolina/1918 (H1), secondary immunization: A/USSR/92/77 (H1), tertiaryimmunization: A/California/04/09 (H1). Three weeks after the finalimmunization and three days prior to generation of hybridomas, mice wereboosted intravenously with 50 μg purified A/Brisbane/59/07-like (H1)virus. B cell hybridomas were produced by methods previously described(see, e.g., de StGroth et al., J Immunol Methods 35: 1-21).

6.7.2 Screening of Hybridoma Supernatants

Hybridoma supernatants were screened for reactivity with A/USSR/92/77(H1) virus and A/California/04/09 (H1) virus by ELISA as described inSection 6.4.1.5. The reactivity of potential clones from one hybridomawith the two H1 viruses are shown in FIG. 34.

6.7.3 Western Blots

To assess binding of cross-reactive anti-H1 monoclonal antibodies,Western blot can be performed. Western blots can be produced by methodsknown in the art (see, e.g., Towbin et al., Proc Natl Acad Sci USA 1979;76(9):4350-4). Samples can be boiled for 5 minutes at 100° C. in loadingbuffer containing SDS and 0.6M DTT. SDS migration buffer can be used forelectrophoresis.

6.7.4 Immunofluorescence Test

To assess binding of cross-reactive anti-H1 monoclonal antibodies,immunofluorescence can be performed. MDCK cells can be infected withvirus at a multiplicity of infection of 1 or 0.5, incubated for 12 hoursat 37° C., and fixed in the absence of trypsin. Infected and uninfectedcells can be incubated with 1 μg/ml mAb for 1 hour at room temperature.Goat anti-mouse fluorescein conjugate (SouthernBiotech) can be used fordetection of mAb binding. The ability of the cross-reactive anti-H1monoclonal antibodies to bind the virus can then be assessed usingimmunofluorescence approaches.

The foregoing is not to be limited in scope by the specific embodimentsdescribed herein. Indeed, various modifications of the antibodies andmethods provided herein and their equivalents, in addition to thosedescribed herein will become apparent to those skilled in the art fromthe foregoing description and accompanying figures. Such modificationsare intended to fall within the scope of the appended claims.

All references cited herein are incorporated herein by reference intheir entirety and for all purposes to the same extent as if eachindividual publication or patent or patent application was specificallyand individually indicated to be incorporated by reference in itsentirety for all purposes.

1.-16. (canceled)
 17. A hybridoma designated 7A7 deposited underprovisions of the Budapest Treaty with the American Type CultureCollection (ATCC, 10801 University Blvd., Manassas, Va. 20110-2209) onMay 22, 2009 (ATCC Accession No. PTA-10058), a hybridoma designated 12D1deposited under provisions of the Budapest Treaty with the American TypeCulture Collection (ATCC, 10801 University Blvd., Manassas, Va.20110-2209) on May 22, 2009 (ATCC Accession No. PTA-10059), a hybridomadesignated 39A4 deposited under provisions of the Budapest Treaty withthe American Type Culture Collection (ATCC, 10801 University Blvd.,Manassas, Va. 20110-2209) on May 22, 2009 (ATCC Accession No.PTA-10060), or a hybridoma designated 66A6 deposited under provisions ofthe Budapest Treaty with the American Type Culture Collection (ATCC,10801 University Blvd., Manassas, Va. 20110-2209) on May 26, 2010 (ATCCAccession No. PTA-11046).
 18. (canceled)
 19. (canceled)
 20. (canceled)21. An isolated monoclonal antibody produced by the hybridoma designated7A7, 12D1, 39A4, or 66A6 of claim
 17. 22. (canceled)
 23. (canceled) 24.(canceled)
 25. A humanized antibody generated from the monoclonalantibody of claim
 21. 26. An isolated antibody that binds to SEQ IDNO:1, SEQ ID NO:124, or SEQ ID NO:125, and neutralizes two or morestrains of an Influenza A virus HA subtype.
 27. (canceled) 28.(canceled)
 29. An isolated antibody that binds to Influenza A virus ofthe H3 subtype, the antibody comprising (a) the variable heavy (VH)domain of the antibody 7A7, 12D1, 39A4, or 66A6; (b) the variable light(VL) domain of the antibody 7A7, 12D1, 39A4, or 66A6; (c) the VH domainof the antibody 7A7, 12D1, 39A4, or 66A6 and the VL domain of theantibody 7A7, 12D1, 39A4, or 66A6; (d) the VH complementaritydetermining regions (CDRs) of the antibody 7A7, 12D1, 39A4, or 66A6; (e)the VL CDRs of the antibody 7A7, 12D1, 39A4, or 66A6; or (f) the VH CDRsof the antibody 7A7, 12D1, 39A4, or 66A6 and the VL CDRs of the antibody7A7, 12D1, 39A4, or 66A6.
 30. A composition comprising the antibody ofclaim
 29. 31. (canceled)
 32. A method of preventing an Influenza virusdisease comprising administering to a subject the antibody of claim 29.33. (canceled)
 34. (canceled)
 35. (canceled)
 36. A method of detecting astrain of Influenza A virus H3 subtype comprising: (a) assaying for thelevel of an Influenza virus HA in cells or a tissue sample of a subjectusing the antibody of claim 29; and (b) comparing the level of theInfluenza virus HA assayed in (a) with the level of the Influenza virusHA in normal tissue samples not infected with Influenza virus, whereinan increase in the assayed level of Influenza virus HA compared to thecontrol level of the Influenza virus antigen is indicative of thepresence of a strain of Influenza A virus H3 subtype.
 37. (canceled) 38.A kit comprising, in a container, the antibody of claim
 29. 39.(canceled)
 40. (canceled)
 41. (canceled)
 42. (canceled)
 43. (canceled)44. (canceled)
 45. (canceled)
 46. An isolated nucleic acid encoding anantibody that binds to Influenza A virus of the H3 subtype, wherein theantibody comprises (a) the amino acid sequence of the VH domain of theantibody 7A7, 12D1, 39A4, or 66A6; (b) the amino acid sequence of the VLdomain of the antibody 7A7, 12D1, 39A4, or 66A6; (c) the amino acidsequence of the VH domain of the antibody 7A7, 12D1, 39A4, or 66A6 andthe VL domain of the antibody 7A7, 12D1, 39A4, or 66A6; (d) the aminoacid sequence of the VH CDRs of the antibody 7A7, 12D1, 39A4, or 66A6;(e) the amino acid sequence of the VL CDRs of the antibody 7A7, 12D1,39A4, or 66A6; or (f) the VH CDRs of the antibody 7A7, 12D1, 39A4, or66A6 and the VL CDRs of the antibody 7A7, 12D1, 39A4, or 66A6. 47.(canceled)
 48. (canceled)
 49. A host cell genetically engineered tocontain or express the nucleic acid of claim
 46. 50. A method ofproducing an antibody, comprising culturing the host cell of claim 49under conditions in which the nucleic acid is expressed and recoveringthe antibody from the host cell culture.